Clavulanic acid amides

ABSTRACT

Amides of the compound of the formula ##STR1## are useful for their β-lactamase inhibitory activity.

The present invention relates to amides of clavulanic acid, to theirpreparation and to compositions containing them. Clavulanic acid of theformula ##STR2## and its salts and esters are produced by the aerobiccultivation of Streptomyces clavuligeris in conventional nutrient mediaat about 25° - 30° C under roughly neutral conditions which pssessβ-lactamase inhibitory and antibacterial activity. Clavulanic acid hasthe following properties:

A. It is a carboxylic acid.

B. It forms a sodium salt which has a characteristic infra-red spectrumsubstantially as shown in Graph 1.

C. It is able to inhibit the growth of strains of Staphylococcus aureus.

D. It is able to synergyze the antibacterial effect of ampicillinagainst β-lactamase producing strains of Escherichia coli, Klebsiellaaerogenes and Staphylococcus aureus.

E. It is able to synergyze the antibacterial effect of cephaloridineagainst the β-lactamase producing strains of Proteus mirabilis andStaphylococcus aureus.

F. It forms a methyl ester which has a molecular weight (by massspectroscopy) of 213,0635 which corresponds to the formula C₉ H₁₁ NO₅.

Thus clavulanic acid may be regarded as a monobasic carboxylic acid ofthe formula C₈ H₉ NO₅ which in the form of its sodium salt has acharacteristic infra-red absorption spectrum substantially as shown inFIG. 1.

The compound produced by Streptomyces clavuligerus which has the aboveproperties has the formula (II): ##STR3##

Thus clavulanic acid may be named3-(β-hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo[3,2,0]heptane-2-carboxylicacid.

The stereochemistry at C₅ and C₂ of the clavulanic acid is the same asthat found in naturally occurring penicillins and cephalosporins so thatclavulanic acid may be represented by the structural formula (I'):##STR4##

Thus a fuller chemical name for clavulanic acid isZ-(2R,5R)-3-(β-hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo[3,2,0]heptane-2-carboxylicacid.

The great usefulness of clavulanic acid may be readily appreciated whenit is realized that certain strains of Klebsiella aerogenes A, thegrowth of which is not inhibited by the prosence of 125 μg/ml. ofampicillin, amoxycillin, carbenicillin or benzyl penicillin or by thepresence of 10 μg/ml. of clavulanic acid, are inhibited by the presenceof less than 12.5 μg/ml. of the previously mentioned penicillins when 5μg/ml. of clavulanic acid is also present. Similar results have beenobserved for combinations containing various esters of clavulanic acid.For example, strains of Klebsiella aerogenes A, the growth of which isnot inhibited by 125 μg/ml. of ampicillin, or by 10 μg/ml of clavulanicacid methyl ester are inhibited by less than 12.5 μg/ml. of ampicillinin the presence of 5 μg/ml. of the clavulanic acid methyl ester. It hasalso been found that strains of Staphylococcus aureus Russell, thegrowth of which is not inhibited by the presence of 100 μg/ml. ofampicillin or by 5 μg/ml of clavulanic acid, are inhibited by thepresence of less than 10 μg/ml. of ampicillin in the presence of 1μg/ml. of clavulanic acid. In tests on female mice, it has been foundthat blood and tissue levels of clavulanic acid considerably in excessof 5 μg/ml. can readily be achieved by subcutaneous administration of100 mg/kg of the sodium salt of clavulanic acid and that useful levelsof clavulanic acid can be obtained after oral administration of 100mg/kg of the sodium salt of clavulanic acid.

Most suitably, the salts of clavulanic acid will be pharmaceuticallyacceptable salts such as the sodium, potassium, calcium, magnesium,aluminium, ammonium and substituted ammonium salts such as thetrimethylammonium, benzathine, procain and like salts conventionallyformed with penicillins or cephalosporins. Non-pharmaceuticallyacceptable salts of clavulanic acid are also included as they are usefulintermediates in the preparation of esters of clavulanic acid, forexample, the lithium or silver salts of clavulanic acid may be reactedwith benzyl bromide to form the useful benzyl ester of clavulanic acid.

Salts of clavulanic acid tend to be more stable tha the parent acid perse and thus form a favoured aspect of this invention. Particularlysuitable salts of clavulanic acid include the sodium and potassium saltswhich have the formula (III') and (IV') respectively: ##STR5##Crystalline forms of such salts may contain water of hydration.

Suitable esters of clavulanic acid include those notionally derived fromalcohols such as methanol, ethanol, propanol, butanol,2,2,2-trichloroethanol, 2,2,2-trifluoroethanol, benzyl alcohol,p-nitrobenzyl alcohol, phenol, acetoxymethanol, pivaloyloxymethol,2-dimethylaminoethanol and other conventional alcohols. Various estersof clavulanic acid are useful intermediates in certain processes for thepurification of clavulanic acid. Many clavulanic acid esters are usefulsynergistic compounds. The activity of such esters might be due tohydrolysis of the ester to the parent acid.

When used herein the term ester includes esters notionally derived froman alcohol or thiol of the formula ROH or RSH where R is an organicresidue. Suitable groups R include alkyl, alkenyl, alkynyl, aryl,arylalkyl or other similar groups any of which may be substituted ifdesired. In order not to increase the molecular weight to anunreasonable extent, groups R do not normally include more than 16carbon atoms, more suitably, not more than 12 carbon atoms and mostsuitably, not more than 8 carbon atoms.

Preferably, the group R is notionally derived from an alcohol ROH or(less favorably) a thiol RSH which is pharmaceutically acceptable.

Suitable substituents which may be included in the group R includehalogen atoms and lower alkoxyl, hydroxyl, lower acyloxyl, loweralkylamino, lower dialkylamino and like groups. The term `lower` meansthat the group contains up to 6 carbon atoms, and preferably up to 4carbon atoms. Thus, for example, R may be a methyl, ethyl, n-propyl,iso-propyl, straight or branched butyl, pentyl, heptyl, octyl, nonyl,decyl, undecyl, dodecyl, vinyl, allyl, butenyl, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, cyclohexenyl, cyclohexadienyl,methylcyclopentyl, methylcyclohexyl, benzyl, benzhydryl, phenylethyl,napthylmethyl, phenyl, naphthyl, propynyl, tolyl, 2-chloroethyl,2,2,2-trichloroethyl, 2,2,2-tri-fluorethyl, acetylmethyl, benzoylmethyl,2-methoxyethyl, 2-dimethylaminoethyl, 2-diethylaminoethyl,2-piperidinoethyl, 2-morpholinoethyl, 3-dimethylaminopropyl,p-chlorobenzyl, p-methoxybenzyl, p-nitrobenzyl, p-bromobenzyl,n-chlorobenzyl, 6-methoxynaphthyl-2-methyl, p-chlorophenyl,p-methoxyphenyl or any like group as well as those groups which areknown from the penicillin or cephalosporin arts to produce esters knownto be readily hydrolyzed in vivo to the parent antibiotic.

Readily hydrolyzable esters include, but are not limited to, those ofthe formulae (V) and (VI): ##STR6## wherein A₁ is a hydrogen atom,alkyl, aryl or aralkyl group; A₂ is a hydrogen atom or methyl group; A₃is an alkyl, aryl or aralkyl groupl X is oxygen or sulphur; Y is oxygenor sulphur and Z is a divalent organic group. Esters of the formulae(V')and (VI') which fairly readily released the clavulanic acid into theblood stream after administration include those wherein A₁ is a hydrogenatom; A₂ is a hydrogen atom or a methyl group and A₃ is a methyl, ethyl,propyl, butyl, benzyl, or phenyl group and those wherein X is oxygen, Yis oxygen and Z is --Ch₂ CH₂ --, --CH:CH---, ##STR7##

When used in conjunction with the preceding formula, the term `alkyl`includes alkyl of up to six carbon atoms; the term `aryl` includesphenyl, naphthyl or phenyl substituted by an inert substituent such as afluorine or chlorine atom or a methyl or methoxyl group or the like;when used herein the term `aralkyl` means an alkyl group substituted byan aryl group.

Particularly suitable esters of the formulae (V) and (VI) include thoseof the formulae (VII) and (VIII): ##STR8## A₄ is a hydrogen atom or amethyl group, A₅ is a methyl, t-butyl or phenyl group and A₆ is ahydrogen atom or a methoxyl group.

Many esters of clavulanic acid differ from analagous esters ofpenicillins or cephalosporins in that they show an enhanced tendency tohydrolyze to clavulanic acid under mild conditions. Thus, for example,simple alkyl esters such as the methyl ester slowly hydrolyze toclavulanic acid in water buffered to pH7. Esters which undergo somehydrolysis under mild conditions are included within the formula (IX):##STR9## wherein R¹ is a hydrocarbon group of 1-9 carbon atomsoptionally substituted by halogen, lower alkoxy, hydroxyl or optionallysalted basic groups of the formula NR² R³ wherein R² is a hydrogen atomor a lower alkyl group, R³ is a hydrogen atom or a lower alkyl group oris attached to R² so that NR² R³ is a 5- or 6- membered ring.

When used witbh reference to formula (IX), the term `lower` means thatthe group contains 1-4 carbon atoms.

Suitably groups R¹ include alkyl and aralkyl groups optionallysubstituted by halogen, methoxyl, hydroxyl or salted NR² R³ groupswherein R² is a methyl or ethyl group and R³ is a methyl or ethyl groupor is joined to R² so that NR² R³ is a pyrrolidine, piperidine ormorpholine group.

Most suitably, alkyl groups R¹ are straight chain groups of up to 6carbon atoms optionally substituted by one methoxyl, hydroxyl, saltedNR² R³ group or one chlorine, bromine or iodine atom or by a CCl₃ or CF₃group.

The esters of clavulanic acid of particular usefulness as synergists arethose which hydrolyze in mammalian tissues, especially human blood, toyield clavulanic acid or a salt thereof because it is believed thatclavulanic acid and its salts tend to be somewhat more usefulantibacterial agents than the esters per se. Many of the esters of theformulae (V)- (VII), (VIII) and (IX) are useful for this purpose.

A further group of paticularly suitable esters are those usefulintermediates which are readily converted to clavulanic acid or a saltthereof by chemical or biochemical techniques which are known from thepenicillin or cephalosporin arts to be sufficiently mild not to degradereactive acid-labile β-lactam rings.

Most suitably, the ester is one removable by hydrogenolysis.Conventional esters for such a process include benzyl, substitutedbenzyl, benzhydryl, substituted benzhydryl, trityl and the like. Thebenzyl ester has proved particularly useful for this purpose.

By and large, the nature of any substitutent in the ester moiety isunimportant as long as it does not interfere with the hydrogenolysisreaction.

The compositions include those in a form adapted for oral, topical orparenteral use and may be used for the treatment of infection in mammalsincluding humans.

Suitable forms of the compositions include tablets, capsules, creams,syrups, suspensions, solutions, reconstitutable powders and sterileforms suitable for injection or infusion. Such compositions may containconventinal pharmaceutically acceptable materials such as diluents,binders, colors, flavors, preservatives, disintegrants and the like inaccordance with conventional pharmaceutical practice in the manner wellunderstood by those skilled in the art of formulating antibiotics.

Injectable or infusable compositions of the clavulanic acid or its saltsare particularly suitable as high tissue levels of the compound ofclavulanic acid can occur after administration by injection or infusion.Thus, one preferred composition aspect comprises clavulanic acid or asalt thereof in sterile form.

Unit dose compositions comprising clavulanic acid or a salt or esterthereof adapted for oral administration form a further preferredcomposition.

Under certain conditions, the effectiveness of oral compositions ofclavulanic acid and its salts and esters can be improved if suchcompositions contain a buffering agent or an enteric coating agent suchthat the compounds of the invention do not have prolonged contact withhighly acidic gastric juice. Such buffered or enterically coatedcompositions may be prepared in accordance with conventionalpharmaceutical practice.

The clavulanic acid or its salt or ester may be present in thecomposition as sole therapeutic agent or it may be present together withother therapeutic agents such as a β-lactam antibiotic. Suitableβ-lactam antibiotics for inclusion in such synergistic compositionsinclude not only those known to be highly susceptible to β-lactamasesbut also those which have a good degree of intrinsic resistance toβ-lactamses. Thus, suitable β-lactam antibiotics for inclusion in thecompositions of this invention include benzylpenicillin,phenoxymethylpenicillin, carbenicillin, methicillin, propicillin,ampicillin, amoxycillin, epicillin, ticarcillin, cyclacillin,6-aminopenicillanic acid, 7-aminocephalosporanic acid,7-aminodesacetoxycephalosporanic acid, cephaloridine, cephalothin,cefazolin, cephalexin, cefoxitin, cephacetrile, cephamandole,cephapirin, cephradine, cephaloglycine and other well known penicillinsand cephalosporins or pro-drugs therefore such as hetacillin,metampicillin, the acetoxymethyl, pivaloyloxymethyl or phthalidyl estersof benzylpenicillin, ampicillin, amoxycillin or cephaloglycine or thephenyl, tolyl or indanyl α-esters of carbenicillin or ticarcillin or thelike.

Naturally, if the penicillin or cephalosporin present in the compositionis not suitable for oral administration then the composition will beadapted for parenteral administration.

When present in a pharmaceutical composition together with a β-lactamantibiotic, the ratio of clavulanic acid or its salt or ester present toβ-lactam antibiotic present may be from, for example, 10:1 to 1:10 andadvantageously may be from 3:1 to 1:3.

The total quantity of antibacterial agents present in any unit dosageform will normally be between 50 and 1500 mg and will usually be between100 and 1000 mg.

Compositions may be used for the treatment of infections of inter alia,the respiratory tract, the urinary tract and soft tissues in humans.

Compositions may also be used to treat infections of domestic animalssuch as mastitis in cattle.

Normally between 50 and 6000 mg of the compositions will be administeredeach day of treatment but more usually between 500 and 300 mg of thecomposition of the invention will be administered per day. However forthe treatment of severe systemic infections or infections ofparticularly intransigent organisms, higher doses may be used inaccordance with clinical practice.

The exact form of the compositions will depend to some extent on themicro-organism which is being treated. For treatment of infections thecompositions of this invention are normally adapted to produce a peakblood level of at least 0.1 μg/ml, more suitably at least 0.25 μg/ml,and preferably at least 1 μg/ml of clavulanic acid.

The penicillin or cephalosporin in synergistic compositions willnormally be present by up to or at approximately the amountconventionally used when that penicillin or cephalosporin is the soletherapeutic agent used in the treatment of infection.

Particularly favoured compositions will contain from 150 - 1000 mg ofamoxycillin, ampicillin or a pro-drug therefore and from 50 - 500 mg ofclavulanic acid or a salt or in vivo hydrolyzable ester thereof and moresuitably from 200 - 500 mg of amoxycillin, ampicillin or a pro-drugtherefore and from 50 - 250 mg of clavulanic acid or a salt or in vivohydrolyzable ester thereof.

The materials present in such compositions may be hydrated if required.The weights of the antibiotics in such composition are expressed on thebasis of antibiotic theroretically available from the composition andnot on the basis of the weight of pro-drug.

A process for the preparation of clavulanic acid and salts and estersthereof is provided which process comprises cultivating a strain of[Streptomyces clavuligerus and recovering clavulanic adic or a saltthereof from the culture medium and thereafter if desired, forming thefree acid or a salt or ester by methods known per se.

Preferably, Streptomyces clavuligerus ATCC 27064 or a high yieldingmutant thereof is used in the process of this invention.

When used herein, the term `cultivation` means the deliberate aerobicgrowth of a clavulanic acid producing organism in the presence ofassimilable sources of carbon, nitrogen and mineral salts. Such aerobicgrowth may take place in a solid or semi-solid nutritive medium, or in aliquid medium in which the nutrients are dissolved or suspended. Thecultivation may take place on an aerobic surface or by submergedculture. The nutritive medium may be composed of complex nutrients ormay be chemically defined. In our hands we have found media containingcomplex nutrients such as yeast extract, soya bean flour and the like tobe particularly suitable.

The nutrient media which may be used for the cultivation of Streptomycesclavuligerus may contain, in the range of 0.1-10% a complex organicnitrogen source such as yeast extract, corn-steep liquor, vegetableprotein, seed protein, hydrolyzates of such proteins, milk proteinhydrolyzates, fish and meat extracts and hydrolyzates such as peptones.Alternatively chemically defined sources of nitrogen may be used such asurea, amides, single or mixtures of common amino acids such as valine,asparagine, glutamic acid, proline and phenylalanine. Carbohydrate(0.1-5%) may be included in the nutrient media but glucose in certainmedia is undesirable having a depressing effect on the yield of thedesired clavulanic acid. Starch or starch hydrolyzates such as dextrin,sucrose, lactose or other sugars or glycerol or glycerol esters may beused. The source of carbon may also be derived from vegetable oils oranimal fats. Carboxylic acids and their salts can be included as asource of carbon for growth and production of β-lactamase inhibitors. Aparticularly suitable low cost medium is one containing soya bean flour(Arkasoy) plus dried malt distillers solubles (Scotasol) plus dextrin.

The addition of antifoam agents such as Pluronic L81 may be necessary tocontrol foaming of certain media in fermenters.

Mineral salts such as NaCl, KCl, MgCl₂, ZnCl₂, FeCl₃, Na₂ SO₄, FeSO₄,MgSO₄ and Na⁺ or K⁺ salts or phosphoric acid may be added to the mediadescribed above particularly if chemically defined; CaCO₃ may be addedas a source of Ca⁺⁺ ions or for its buffering action. Salts of traceelements such as nickel, cobalt or manganese may also be included.Vitamins may be added if desired.

When used herein the term `mutant` includes any mutant strain whicharises spontaneously or through the effect of an external agent whetherthat agent is applied deliberately or otherwise. Suitable methods ofproducing mutant strains include those outlined by H. I. Adler inTechniques for the Development of Micro-Organisms in `Radiation andRadioisotopes for Industrial Micro-Organisms` , Proceedings of aSymposium, Vienna, 1973, page 241, International Atomic Energy Authorityand these include:

i. Ionizing radiation (such s X- and γ-rays), uv light, uv light plus aphotosensitizing agent (such as 8-methoxypsoralen), nitrous acid,hydroxylamine, pyrimidine base analogues (such as 5-bromouracil),acridines, alkylating agents (such as mustard gas, ethyl-methanesulphonate), hydrogen peroxide, phenols, formaldehyde, heat, and

ii. Genetic techniques such as recombination, transformation,transduction, lysogenization, lysogenic conversion and selectivetechniques for spontaneous mutants.

Cultivation of Streptomyces clavuligerus normally takes place in thetemperature range 15°-40° C, usually 20°-35° C and prefrably, 25°-30° Cand at a pH of between 5 and 8.5, preferably between 6 and 7.5.

The Streptomyces clavuligerus may be cultivated in the above media inglass conical flasks aerated by shaking on a rotary shaker or in baffledstainless steel fermenters stirred with vaned disc impellers and aeratedwith a sparger. The fermentation may also be carried out in a continuousfashion.

The starting pH of the fermentation is typically 7.0 and maximum yieldof clavulanic acid obtained in 2-10 days at 20°-35° C. In a stirredstainless steel fermenter using the Arkasoy/Scotasol/Dextrin mediumdescribed above the preferred temperature is 26° C and peak yieldsclavulanic are obtained within 5 days.

Clavulanic acid may be extracted from culture filtrate by a variety ofmethods. Solvent extraction from cold culture filtrate adjusted to acidpH values and methods based on the anionic nature of the metabolite suchas the use of anion exchange resins have been found to be particularlyuseful. The cells of the Streptomyces clavuligerus are normally firstremoved from the fermentation by filtration or centrifugation beforesuch extraction procedures are commenced.

In the solvent extraction process, the culture filtrate is chilled andthe pH lowered into the region of pH 2-3 by the addition of acid whilethoroughly mixing with a water immiscible organic solvent such asn-butylacetate, methylisobutylketone, n-butanol or ethylacetate. Theacid used to lower the pH of the medium is normally a mineral acid suchas hydrochloric, sulphuric, nitric, phosphoric or the like acid.n-Butanol is a particularly suitable solvent for use in the extractionof the acidified culture filtrate. After separation of the phases bycentrifugation, the β-lactamase inhibiting metabolite is back extractedfrom the solvent phase into aqueous sodium bicarbonate or potassiumhydrogen phosphate buffer, CaCO₃ suspension or water while maintainingthe pH at approximate neutrality, for example, at pH 7.0. This aqueousextract after separation of phases may be concentrated under reducedpressure and freeze dried to give a crude preparation of a salt ofclavulanic acid. This preparation is stable when stored as a dry solidat -20° C.

In the anion exchange resin process, the clarified culture filtrate atan approximately neutral or slightly acid pH, for example pH 6-7, ispercolated down a column of weak or strong base anion exchange resinsuch as Amberlite IR4B or Zerolit FFIF respectively until the resin issaturated and the β-lactamase inhibiting material emerges from thebottom. The column is then washed with water and eluted with aqueoussodium chloride. The β-lactamase inhibiting fractions are collected,bulked, desalted and freeze dried to yield a crude solid salt ofclavulanic acid.

An alternative form of the extraction process is to contact the culturefiltrate (usually at approximately neutral pH) containing a salt ofclavulanic acid, with an organic phase in which is dissolved a waterinsoluble amine. Suitable organic solvents include such conventionalwater immiscible polar solvents as methylisobutylketone.trichloroethylene and the like. Suitable amines include secondary ortertiary amines in which one of the substituent groups is a long chainaliphatic group, for xample, of 12-16 carbon atoms and the other is atertiaryalkyl group so that the molecule is lipophilic. Amberlite LA2has proved a successful amine. Normally the amine is used as its acidaddition salt.

After this extraction process the clavulanic acid is present in theorganic phase as the amine salt. The organic phase is then separatedfrom the culture filtrate. The clavulanic acid may be back extractedinto an aqueous phase by back extraction with a salt solution,preferably a concentrated solution of sodium chloride, sodium nitrate orthe like. The crude salt of clavulanic acid may then be obtained byfreeze drying or the like.

Other primary methods of isolation which may be used includeconventional methods such as adsorption onto carbon, ion pairextraction, precipitation, salting out and molecular filtration butthese methods are not usually as successful as the above describedmethods which are preferred.

Further purification of the crude solids obtained by methods describedabove may be obtained by a variety of methods but ion exchange columnchromatography is particularly suitable especially when using Isopor,De-Acidite FFIP SRA64 or DEAE cellulose. The DeAcidite column isgradient eluted with aqueous solution of a salt and such sodium chloride(0-0.5M). The column of DEAE cellulose in 0.01M phosphate buffer at pH7is eluted with a salt solution, normally a NaCl solution (0-0.2M NaCl in0.01M phosphate buffer pH7). Active fractions may be detected by theirβ-lactamase inhibitory activity and their antibacterial acitivityagainst Klebsiella aerogenes in an agar diffusion assay. The fractionscontaining the bulk of this activity are then combined and concentratedto a small volume under vacuum. This crude preparation of the clavulanicacid salt is desalted by percolating down a column of Bio Gel P2. Theactive desalted material is then concentrated, mixed with ethanol andfurther chromatographed on a cellulose column usingbutanol/ethanol/water 4/1/5 v/v top phase, as solvent.

Fractions containing material which inhibited Escherichia coliβ-lactamase are bulked, evaporated to dryness under vacuum, redissolvedin water and freeze-dried to give a salt of clavulanic acid as a whitesolid.

The methods we have found most useful in detecting clavulanic acid inculture filtrate are paper chromatography and a bioautographic detectionsystem. Clavulanic acid may be assayed by making use of its β-lactamaseinhibiting activity. Thin layer chromatography may be used to detectclavulanic acid in solid preparations. These detection and assayprocedures are described hereinafter.

A variation of the process for the preparation of a pure form ofclavulanic acid or its salts comprises isolating an impure form ofclavulanic acid or alt thereof, forming an ester of clavulanic inconventional manner, purifying the ester and thereafter regeneratingclavulanic acid or a salt thereof from the ester.

The impure clavulanic acid or its salts used in this process willnormally contain at least 1% by weight of the antibiotic.

Suitable esters for use in this process include those which may becleaved by hydrogenolysis, enzymatic methods or by hydrolysis under verymild conditions.

One suitable group of esters used in this process is that of the formula(X): ##STR10## wherein A₇ is a hydrogen atom or an optionallysubstituted phenyl group and A₈ is an optionally substituted phenylgroup.

Most suitably A₇ is a hydrogen atom or a phenyl, tolyl, chlorophenyl ormethoxyphenyl group and A₈ is a phenyl, tolyl, chlorophenyl ormethoxyphenyl group.

Preferably A₇ is a hydrogen atom and A₈ is a phenyl group.

The esters of formula (X) may be cleaved by hydrogenolysis to yieldclavulanic acid or a salt thereof.

Other groups of esters which may be used in this process include thoseof formulae (V) and (VI) as hereinbefore described. Such esters may beconverted to salts of clavulanic acid by mild alkaline hydrolysis, forexample, at pH 7.5.

The impure form of clavulanic acid or salt thereof which is to bepurified in the process may be in the form of a solid or solution whichwill usually also contain considerable quantities of organic orinorganic impurities.

The clavulanic acid or salt thereof may be converted into an ester bythe esterification reactions referred to hereinafter. The preferredmethod of forming the required ester of clavulanic acid is by thereaction of a salt of clavulanic acid with an esterifying agent such asa reactive halide, sulphonate ester or the like as hereinafterdescribed. Such reactions are frequently carried out in an organicsolvent of high dielectric constant such as dimethylformamide,dimethylformamide/acetone, dimethylsulphoxide, N-methylacetamide,hexamethylphosphoramide and the like.

If desired, the salt of clavulanic acid may be dissolved in the solventin conventional manner or it may be bound to a polymeric support.Suitable supports for use in this process include strong base anionexchange resins, especially those possessing a macroreticular naturewhich permits the use of non-aqueous solvent systems. Amerblyst A26 issuitable for this purpose. The clavulanic acid salt may be adsorbed ontothe resin from the culture filtrate and the resin then suspended indimethylformamide containing sodium iodide or alternatively elutedcolumnwise with a solution of sodium iodide in diemthylformamide or in amixture of dimethylformamide and acetone.

Once formed, the impure ester of clavulanic acid is normally purifiedchromatographically. In such procedures the ester is normally dissolvedin an organic solvent such as ethylacetate, methylene chloride,chloroform, cyclohexane or similar solvents. The solid phase used in thechromatographic process is normally an inert material such as silica gelor chromatographically similar materials.

The fractions emerging from the column may be tested for the presence ofthe clavulanic acid by making use of its synergistic properties. Activefractions are normally combined and the organic solvent evaporated offunder reduced pressure.

The ester resulting from this process is generally of acceptable purity,but the material may be rechromtographed if desired.

This purified ester of clavulanic acid may be converted to clavulanicacid or a salt thereof by the before mentioned methods.

A particularly suitable method of obtaining clavulanic acid or its saltis by hydrogenation of a compound of the formula (X) as hereinbeforedescribed. Such reactions normally take place in the presence of atransition metal catalyst using low or medium pressures of hydrogen. Thereaction may be carried out at high, ambient or depressed temperatures,for example at 0°-100° C. Particularly suitble reaction conditions forsuch hydrogenations will use a slightly superatmospheric pressure ofhydrogen at an approximately ambient (12°-20° C) temperature. Thereaction may be carried out in conventional solvents such as loweralkanols, for example, ethanol. We have found that a particularlysuitable catalyst is palladium on charcoal.

If the hydrogenation is carried out in the presence of a base, then asalt of clavulanic acid is produced; for example, the sodium orpotassium salts result if the reaction is carried out in the presence ofsodium or potassium hydrogen carbonate.

The clavulanic acid or salt thereof resulting from such reactions isgenerally of good purity.

Esters or clavulanic acid may be prepared by the esterification ofclavulanic acid or a salt thereof by conventional methods.

Suitable methods of ester formulation include (a) reaction of a salt ofthe acid of clavulanic acid with a compound of the formula Q--R where Qis a readily displaceable group and R is an organic group; (b) thereaction of clavulanic acid with a diazolkane and (c) the reaction ofclavulanic acid with an alcohol ROH in the presence of a condensationpromoting agent such as carbodiimide or the like.

Suitable salts of clavulanic acid which may be reacted with compoundsR--Q include alkali metal salts such as the sodium or potassium salts orother conventional salts such as the silver salt.

Suitable groups Q include those atoms or groups known to be displaceableby carboxylate anions and include chlorine, bromine and iodine atoms,sulphonic acid esters such as the O.SO₂ CH₃ or O.SO₂ C₆ H₄ CH₃ groups,active ester groups such as the 0.CO.H or O.CO.CF₃ group and otherconventional groups displaceable by nucleophiles.

The preceding reaction is normally carried out in an organic solvent ofrelatively high dielectric constant such as dimethylformamide, acetone,dioxane, tetrahydrofuran or the like and at a non-extreme temperaturesuch a -5° C to 100° C, more usually +5° C to 30° C, for example atambient temperature.

The reaction of clavulanic acid with a diazoalkane is a mild method ofmaking alkyl, aralkyl or similar esters. The diazotization reaction maybe performed under conventional reaction conditions, for example at anon-extreme temperature and in a conventional solvent. Such reactionsare normally carried out at between about -5° C and 100° C, more usuallyfrom 5° C to 30° C, for example at ambient temperature. Suitablesolvents for this reaction include lower alkanols such as methanol andethanol and solvents such as tetrahydrofuran, dioxane and the like.Ethanol has proved a particularly useful solvent for this reaction.

The reaction of clavulanic acid with an alcohol in the presence of acondensation promoting agent will normally take place in an inertorganic solvent of relatively high dielectric constant such asacetonitrile. This reaction is usually carried out at an ambient ordepressed temperature, for example at -10° C to +22° C, more usually -5°C to +18° C, for example initially at 0° C and thereafter graduallywarming to about 15° C. The condensation promoting agent used isnormally one which removes water from the reaction mixture. Suitableagents include carbodiimides, carbodiimidazoles or equivalent reagents.Dicyclohexylcarbodiimide has proved to be a particularly suitablecondensation promoting agent for use in this process. In order tominimize self-condensation of the clavulanic acid, this reaction isusually carried out in the presence of a considerable excess of thealcohol.

Other suitable methods of ester formation include (d) removal of theelements of carbon dioxide from a compound of the formula (XI) ##STR11##wherein R⁴ is an inert organic group; and (e) reaction of a compound ofthe formula (XI) with an alcohol ROH (or less favorably with a thiolRSH).

The elements of carbon dioxide may be removed from the compound offormula (XI) spontaneously during its preparation or, alternatively, byheating the compound of the formula (XI) in an inert solvent. Suitableinert solvents include ether solvents such as diethylether,tetrahydrofuran, dioxane, and the like. In many cases the compound ofthe formula (XI) decomposes spontaneously even at a depressedtemperature, for example, at -5° C, to yield an ester of the formula##STR12## wherein R⁴ is an inert group within the definition of R.

When the compound of the formula (XI) is to be reacted with an alcohol(or less favorably with a thiol) then this reaction is normally carriedout in an inert solvent such as an ether solvent in the presence of anexcess of the alcohol (or thiol) in order to prevent self-condensationof the clavulanic acid derivative.

The methods of esterification are not, in general, as useful as thoseinvolving reaction of a salt of clavulanic acid with R-C1 ashereinbefore described.

The compound of the formula (XI) may be prepared by the reaction of asalt of clavulanic acid with Cl.CO.O.R⁴ or the chemical equivalentthereof. Normally this reaction is carried out at a depressedtemperature, for example, at a temperature not greater than 5° C, and inan inert solvent, for example diethylether, tetrahydrofuran, dioxane andthe like. Most suitably the salt of clavulanic acid used in thisreaction is a lipophilic salt so that it will dissolve in the solventalthough if desired the less favorable sodium salt may be employed bysuspending it in the reaction medium.

DESCRIPTION 1 Assay Suitable For Detection Of Clavulanic Acid

Principle of the Assay

Solutions containing clavulanic acid (culture filtrate, samples fromisolation procedure and the like) are incubated for 15 minutes with aβ-lactamase preparation in 0.05M phosphate buffer at pH 7 and 37° C.During this time, enzyme inhibition or inactivation occurs. Substrate(benzylpenicillin) is then added and incubation continued for 30 minutesat 37° C. The amount of enzymic degradation of the substrate topenicilloic acid is determined by the hydroxylamine assay forpenicillin. The amount of β-lactamase used is such as to give 75%hydrolysis of the benzylpenicillin in 30 minutes at 37° C.

The extent of hydrolysis is a reflection of the amount of enzymeremaining uninhibited. The results are expressed as percent inhibitionof the enzyme activity by a given dilution of the clavulanicacid--containing solution (e.g. culture filtrate) or the concentrationof clavulanic acid (αg/ml giving 50% inhibition of the enzyme under theabove stated conditions (I₅₀).

β-lactamase Enzyme

The β-lactamase produced by Escherichia coli JT4 is used as an enzyme.This culture is an ampicillin resistant strain and owes its resistanceto the production of an R-factor controlled β-lactamase. Other similarR-factor controlled β-lactamases may be used if desired.

The culture maintained on nutrient agar slopes, is inoculated into 400ml. of sterile Tryptone medium contained in a 2 liter conical flask.This medium has the following composition Tryptone (Oxoid) 32 g/l, yeastextract (Oxoid) 20 g/l, NaCl 5 g/l and CaCl₂ 6H₂ O 2..2 g/l. The finalpH was adjusted to 7.4 with dilute NaOH. The flask is shaken at 25° Cfor 20 hours on a rotary shaker at 240 r.p.m.

The bacterial cells are collected by centrifugation, washed with 0.05Mphosphate buffer pH 7 (resuspended and centrifuged) and resuspended inwater to give cell concentration 25 times that in the cultivationmedium. This cell suspension was then disrupted in an MSE ultrasonicdisintegrator at 4° C. The cell debris was removed by centrifugation andaliquots of the supernatant stored deep frozen. For use in the assayprocedure, the supernatant is diluted in 0.005M phosphate buffer untilit gives about 75% hydrolysis of a 1 mg/ml. solution of benzylpenicillinin 30 minutes at 37° C.

Assay Procedure

Suitable dilutions of the inhibitor preparation and β-lactamase solutionare mixed and incubated at 37° C for 15 minutes (Test). A control withbuffer in place of inhibitor preparation is also incubated.Benzylpenicillin solution (substrate) is then added to test and controlmixtures, incubation continued for a further 30 minutes at 37° C. Theresidual benzylpenicillin in each mixture is then estimated using thehydroxylamine assay as described by Batchelor et al, Proc. Roy. Soc., B154, 498 (1961). 6 ml. of hydroxylamine reagent are added to all tests,controls and blanks and are allowed to react for 10 minutes at roomtemperature prior to the addition of 2 ml of ferric ammonium sulphatereagent. The absorption of the final solutions is measured in an E.E.L.Colorimeter or a Spectrophotometer at 490 nm against the reagent blank.The composition of the reactions, tests and blanks prior to thehydroxylamine assay are as follows:

    ______________________________________                                        Components             Benzyl-                                                (all dissolved in or   penicillin                                                                              Con- Reagent                                 diluted with 0.005M    Blank     trol Blank                                   pH 7 phosphate buffer)                                                                       Test    ml.       ml.  ml.                                     ______________________________________                                        Escherichia coli                                                              β-lactamase solution                                                                    1.9     0.0       1.9  1.9                                     Inhibitor solution                                                                           0.1     0.0       0.0  0.0                                     Benzylpenicillin                                                              5mg/ml.        0.5     0.5       0.5  0.0                                     0.005M pH 7 phosphate                                                         buffer         0.0     2.0       0.1  0.6                                     ______________________________________                                    

Calculation of Results

The percentage inhibition of the β-lactamase is calculated as follows

Absorption of benzylpenicillin blank minus absorption of control(uninhibited reaction) = x

Absorption of test (inhibited reaction) minus absorption of control(uninhibited reaction) = y

    % inhibition = (y/x) × 100

To obtain the I₅₀ value, the inhibitor preparation is diluted until 50%inhibition of the β-lactamase inactivation of benzylpenicillin isobtained in the above procedure.

DESCRIPTION 2 Paper Chromatographic Detection Of Clavulanic Acid

Culture filtrate and a reference solution of clavulanic acid (250 μg/mlpartially purified preparation), are spotted (20 μl/origin) onto WhatmanNo. 1 paper strips 1 cm. wide. The chromatograms are run by descendingchromatography for 16 hours at 5° C using n-butanol/isopropanol/water,7/7/6 v/v as solvent. The strips are dried at 40° C and laid on agarplates containing 6 μg/ml benzylpenicillin and seeded with a β-lactamaseproducing strain of Klebsiella aerogenes (synergism system). The platesare incubated overnight at 30° C and clavulanic acid revealed as a zoneof inhibited growth. The R_(f) value of the zone was 0.46. The 6 μg/mlbenzylpenicillin alone is below the concentration required to kill theKlebsiella aerogenes but in the presence of a β-lactamase inhibitor,this concentration becomes toxic, that is to say there is synergism.

Use of the above synergism system enables clavulanic acid to be detectedat concentrations below those at which it shows antibacterial activity.

DESCRIPTION 3 Thin Layer Chromatographic Detection Of Clavulanic AcidSodium Salt

Solutions of clavulanic acid sodium salt preparations are spotted (5 μlof lmg/ml) onto glass plates coated with a 0.25 mm layer of silica gel(F254) as supplied by E. Merck, Darmstadt, Germany. The chromatogramsare run at 22° C using the top phase of the mixturen-butanol/ethanol/water 4/1/5 v/v. The chromatogram plates are dried at40° C and clavulanic acid sodium salt located by bioautography on agarplates containing 6 μg/ml. benzylpenicillin and seeded with Klebsiellaaerogenes (synergism system -- see section on paper chromatographyabove). The agar surface is covered by a fine filter cloth before layingthe TLC plate onto it. After allowing 15-30 minutes for wetting anddiffusion, the TLC plate is lifted off with the aid of the filter clothand the agar plate incubated overnight at 30° C to reveal the zones ofinhibited growth. The R_(f) value of clavulanic acid sodium salt in theabove solvent is approximately 0.37. Two spray reagents, Ehrlich andtriphenyltetrazolium chloride are also used to reveal the clavulanicacid sodium salt zone. The former reagent consists of 300 mg ofp-dimethylaminobenzaldehyde dissolved in 9 ml. of ethyl alcohol, 54 ml.of n-butanol and 9 ml of concentrated HCl. On heating the sprayed TLCplate at 120° C for 1-2 minutes, clavulanic acid sodium salt appears asa pink spot. The triphenyltetrazolium chloride reagent consists of amixture of 1 volume of a 4% solution of this compound in methanol with 1volume of methanolic sodium hydroxide. After spraying, the TLC platesare heated at 80° C. Clavulanic acid sodium salt appears as a red spoton a white background.

The following Examples are illustrative:

EXAMPLE 1 Cultivation of Streptomyces clavuligerus

Streptomyces clavuligerus was cultivated at 26° C on agar slopescontaining 1% Yeatex (yeast extract), 1% glucose and 2% Oxoid agar No.3, pH 6.8. A sterile loop was used to transfer mycelium and spores fromthe slope into 100 ml of a liquid medium in a 500 ml Ehrlenmeyer flask.The liquid medium had the following composition:

Oxoid Malt Extract: 10 g/l

Oxoid Bacteriological Peptone: 10 g/l

Glycerol: 20 g/l

Tap water: 1 liter

The medium was adjusted to pH 7.0 with sodium hydroxide solution and 100ml. volumes dispensed into flasks which were closed with foam plugsprior to autoclaving at 15 lb/sq.in. for 20 minutes. An inoculated seedflask was shaken for 3 days at 26° C on a rotary shaker with 2 inchthrow and a speed of 240 r.p.m. Production stage flasks containing theliquid medium described above were inoculated with 5% vegetativeinoculum and grown under the same conditions as the seed flask. Samplesof culture filtrate were assayed for inhibitor action against theβ-lactamase of Escherichia coli JT4. Optimum activity was obtained after3 days. The results are shown in Table 1. A zone of clavulanic acid atR_(f) 0.46 was seen when the culture filtrate was examined by the paperchromatographic method previously described. The increase in size of thezone paralleled the increase in the β-lactamase inhibitor assay.

Streptomyces clavuligerus was also cultivated in 2 liter shaken flaskscontaining 400 mls. of medium (Production stage) using the same mediumand cultural conditions as described earlier in this Example. In theselarger vessels, growth of the organism was slower and optimumβ-lactamase inhibitory activity was achieved 7-9 days after inoculationwith the vegetative seed. The results are also shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        β-Lactamase Inhibiting Activity                                          of Streptomyces clavuligerus                                                  Grown in 500 ml. and                                                          2000 ml. Flacks                                                               Fermen- % Inhibition of Escherichia coli                                      tation  β-lactamase at a final dilution of                               Time    1/2500 of culture filtrate                                            (Days)  500 ml. Shaken Flask                                                                          2000 ml. Shaken Flask                                 ______________________________________                                        1       15              --                                                    2       30              --                                                    3       55              --                                                    4       50              10                                                    5       51              21                                                    6       57              36                                                    7       --              51                                                    8       --              53                                                    9       --              50                                                    ______________________________________                                    

EXAMPLE 2 Cultivation of Streptomyces clavuligerus

A seed flask prepared as in Example 1 was used to inoculate 500 ml.conical flasks containing 100 ml. aliquots of the following medium indeionized water:

Soluble Starch: 2% w/v

Glycerol: 0.3% w/v

Scotasol: 0.1% w/v

Arkasoy: 1% w/v

FeSO₄.7H₂ O 0.01% w/v

The medium was sterilized by autoclaving at 15 p.s.i. for 20 minutes andinoculated by the addition of the 5% vegetative seed stage. The flaskswere shaken at 26° C on a rotary shaker as in Example 1. Optimum titreof clavulanic acid was achieved between 3-5 days. A dilution of 1/2500of the culture filtrate gave 60% inhibition in the β-lactamaseinhibition assay. A zone of clavulanic acid was seen at R_(f) 0.46 whenusing the paper chromatographic (bioautographic) method previouslydescribed. This zone increased in size in parallel with the increase ofthe activity in the β-lactamase inhibitor assay.

[Soluble starch supplied by British Drug Houses Ltd., Poole, U.K.;

Scotasol is dried distillers solubles supplied by Thomas Borthwich Ltd.,60 Wellington Street, Glasgow, UK;

Arkasoy is soya bean flour supplied by British Arkady Co., Old Trafford,Manchester, UK].

EXAMPLE 3 Cultivation of Streptomyces clavuligerus

A seed flask as produced in Example 1 was used to inoculate 500 ml.conical flasks containing 100 ml aliquots of the following mediumprepared in deionized water and sterilized as previously described. Theinoculum level was 5%.

Dextrin: 2% w/v

Arkasoy: 1% w/v

Scotasol: 0.1% w/v

FeSo₄ 7H₂ O: 0.01% w/v

The inoculated flasks were shaken at 26° C. Optimum β-lactamaseinhibitory activity was achieved between 3-5 days. The activity wassimilar to that achieved in Example 2.

[Dextrin is supplied by C P C (UK) Ltd., Trafford Park, Manchester, UK]

EXAMPLE 4 Cultivation of Streptomyces clavuligerus

The seed stage as described in Example 1 was used to inoculate 500 ml.conical flasks containing the following medium prepared in deionizedwater.

Dextrose: 1% w/v

Soyabean Meal: 1% w/v

Scotasol: 0.05% w/v

CaCO₃ : 1% w/v

These flasks were treated exactly as in previous Examples and culturedunder identical conditions. β-lactamase inhibitory activity was producedbetween 3-5 days. Culture filtrate at a final dilution of 1/2500 gave35-45% inhibition in the β-lactamase inhibition assay.

EXAMPLE 5 Cultivation of Streptomyces clavuligerus

β-lactamase inhibitory activity attributable to clavulanic acid wasproduced using the following medium with identical seed stage andcultivation conditions to Example 1.

Glycerol: 2% w/v

Soyabean Meal: 1.5% w/v

MgSO₄ : 0.1% w/v

K₂ hpo₄ : 0.1% w/v

Medium prepared in deionized water

β-lactamase inhibitory activity reached a maximum level between 3-5 daysand was of a similar order to that produced in Example 4.

EXAMPLE 6 Cultivation of Streptomyces clavuligerus

The following medium produced clavulanic acid when using the conditionsand vegetative seed inoculum as described in Example 1.

Glucose: 2%

Lab Lemco (Oxoid): 1%

Oxoid Yeast Extract: 0.3%

CaCO₃ : 0.3%

Medium prepared in deionized water.

Optimum titres were achieved in 3-5 days and a 1/2500 dilution of theculture filtrate gave 35-45% inhibition in the β-lactamase enzymeinhibiton assay.

EXAMPLE 7 Cultivation of Streptomyces clavuligerus

As in Examples 4, 5 and 6 the following medium produced 35-45%inhibition (1/2500 dilution) in the β-lactamase assay at the optimumtitre which is reached 3-5 days after inoculation. All conditions wereas previously described.

Glucose: 2% w/v

Arkasoy: 1% w/v

CaCO₃ : 0.02% w/v

CoCl₂.6H₂ O: 0.0001% w/v

Medium prepared in deionized water

EXAMPLE 8 Cultivation of Streptomyces clavuligerus

The following production stage medium when used under standardcultivation conditions as described in previous Examples produced 20-30%inhibition at 1/2500 dilution in the β-lactamase assay between 3-5 daysafter inoculation. Using the paper chromatographic method previouslydescribed, a zone of clavulanic acid was seen at R_(f) 0.46 when culturefiltrate was examined.

Scotasol: 2%

Oxoid Yeast Extract: 1%

Medium prepared in tap water

Final pH 7.0

EXAMPLE 9 Cultivation of Streptomyces clavuligerus

Under standard cultivation conditions, the following medium producedclavulanic acid 3-5 days after inoculation with the vegetative seed. A1/2500 dilution of the culture gave 20-30% inhibition in the β-lactamaseinhibition assay.

    ______________________________________                                                               g/l                                                    ______________________________________                                        Glycerol                 15                                                   Sucrose                  20                                                   Proline                  2.5                                                  Monosodium Glutamate     1.5                                                  NaCl                     5.0                                                  K.sub.2 HPO.sub.4        2.0                                                  CaCl.sub.2               0.4                                                  MnCl.sub.2 4H.sub.2 O    0.1                                                  FeCl.sub.3 6H.sub.2 O    0.1                                                  ZnCl.sub.2               0.05                                                 MgSO.sub.4 7H.sub.2 O    1.0                                                  Medium prepared in deionized water                                            Final pH 7.1                                                                  ______________________________________                                    

EXAMPLE 10 Cultivation of Streptomyces clavuligerus

A stock Yeatex/glucose agar slope was used to inoculate a Yeatex/glucoseagar slope in a Roux bottle by making a mycelium/spore suspension insterile water. The Roux bottle slope was incubated at 26° C for 10 days.To this slope 100 mls. of sterile water was added and a mycelialsuspension prepared. This was used to inoculate 50 liter of steamsterilized seed medium of the following composition in tap water.

    ______________________________________                                        Oxoid Malt Extract      1% w/v                                                Oxoid Bacteriological Peptone                                                                         1% w/v                                                Glycerol                1% w/v                                                10% Pluronic L81 Antifoam in                                                  Soybean Oil             0.05% w/v                                             ______________________________________                                    

[Pluronic supplied by Jacobs and Van den Berg UK Ltd., 231 The Vale,London, W3 containing a polypropylene-polyethylene block polymer, andSoyabean Oil supplied by British Oil and Cake Mills Ltd., StoneferryRoad, Hull, U.K.] .

the medium was contained in a 90 liter stainless steel baffledfermenter, agitated by a 5 inch vaned disc impeller at 240 r.p.m.Sterile air was supplied at 50 l/min and the tank incubated at 26° C.

After 72 hours, the seed fermenter was used to inoculate 150 liter ofthe same medium using a 5% v/v addition by sterile transfer. Thisproduction stage medium was contained in a 300 L stainless steel, fullybaffled fermenter agitated by a 81/2 inch vaned disc impeller at 210r.p.m. Sterile air was supplied at 150 l/min. The fermentation wasmaintained at 26° C. Antifoam was added when required in 10 ml. shots(10% Pluronic L81 in soyabean oil). Samples were removed for β-lactamaseinhibition assay at regular intervals. The fermenter was harvestedbetween 4-5 days at the optimum level of β-lactamase inhibitory activity(Table 2).

                  TABLE 2                                                         ______________________________________                                        β-Lactamase Inhibitory Activity of Samples of                            Culture Filtrate taken from a 300 litre                                       Fermentation of Streptomyces Clavuligerus                                     Fermentation % Inhibition in β-lactamase                                 Time         Inhibition Assay at a Final                                      (days)       Dilution of 1/2500                                               ______________________________________                                        1.0          12                                                               1.5          20                                                               2.0          31                                                               2.5          36                                                               3.0          50                                                               3.5          54                                                               4.0          51                                                               4.5          56                                                               5.0          55                                                               ______________________________________                                    

EXAMPLE 11 Cultivation of Streptomyces clavuligerus

The seed fermenter was run exactly as described in Example 10 using thesame medium.

After 72 hours, the seed fermenter was used to give a 5% v/v vegetativeinoculum into a 300 liter stainless steel fully baffled fermentercontaining 150 liter of steam sterilized medium agitated by an 81/2 inchvaned disc impeller at 210 r.p.m. Sterile air was supplied at 150 l/min.The fermentation was maintained at 26° C. Antifoam was added whenrequired in 10 ml. shots (10% Pluronic L81 in soya bean oil).

The medium used in the production stage was as described in Example 3with the addition of 0.05% v/v of 10% Pluronic L81/soyabean oil antifoamprior to sterilization.

The β-lactamase inhibitory activity of fermentation samples was similarto those of Example 10 (see Table 2). Paper chromatographic examinationrevealed a zone of clavulanic acid at R_(f) 0.46 using thebioautographic (synergism) method previously described. The size of theclavulanic acid zone incresed in parallel with the increase in theβ-lactamase inhibitor assay.

EXAMPLE 12 Cultivation of Streptymyces clavuligerus

100 mls of sterile water was added to a sporing culture which had beengrown on Bennetts agar in a Roux bottle for 10 days at 26° C. Amycelium/spore suspension was produced and used to inoculate 75 litersof steam sterilized medium of the following composition in tap water.

Dextrin: 2% W/V

Arkasoy `50`: 1% W/V

10% pluronic L81 in soyabean oil: 0.03% V/V

The pH of the medium was adjusted to 7.0

The medium was contained in a 100 liter stainless steel baffledfermenter, agitated by a 71/2 inch vaned disc impeller at 140 rpm.Sterile air was supplied at 75 l/minute and the tank incubated for 72hours at 26° C.

The contents of the seed fermenter were used to inoculate 1500 liters ofsteam sterilized medium of the following composition in tap water.

Arkasoy `50`: 1.5% W/V

Glycerol: 1.0% W/V

Kh₂ po₄ : 0.1% w/v

10% pluronic L81 in soyabean oil: 0.2% V/V

The pH of the medium was adjusted to 7.0

The medium was contained in a 2000 liter stainless steel fully baffledfermenter agitated by two 19" vaned disc impellers at 106 r.p.m.

Sterile air was supplied at 1200 liters per minute. Antifoam was addedin 25 ml amounts as required. (10% Pluronic L81 in soyabean oil). Thefermentation was controlled at 26° C until a maximum yield of clavulanicacid was obtained between 3-5 days when 200-300 μg/ml of clavulanic acidwere produced.

EXAMPLE 13 Cultivation of Streptomyces clavuligerus

Inoculum was produced in a seed flask as previously described, but usingthe medium described in Example 3 (with pH of the medium adjusted to7.0). This was used to inoculate 500 ml conical flasks containing 100 mlaliquots of the following medium prepared in deionized water andsterilized. The inoculum level was 5%.

Prichem P224: 1% W/V

Arkasoy `50`: 1.5% W/V

Kh₂ po₄ : 0.1% w/v

the pH of the medium was adjusted to 7.0

The inoculated flasks were shaken at 26° C and optimum β-lactamaseinhibitory activity was achieved between 3-5 days. Levels of 300- 500μg/ml of clavulanic acid were achieved.

Prichem P224 is a triglyceride supplied by Prices Limited, Bromborough,Bebington, Wirral, Cheshire, U.K.

EXAMPLE 14 Isolation of Crude Clavulanic Acid Sodium Salt

Harvested culture liquor produced as described in Example 10 wasclarified by continuous-flow centrifugation and the mycelium discarded.From 150 liter of fermentation liquor 120 liter of clarified culturefluid was obtained. This filtrate gave 58% inhibition in the β-lactamaseinhibition assay at 1/2500. The filtrate was chilled to 5° C and 40liter of n-butanol added. The mixture was stirred and 25% H₂ SO₄ addeduntil the pH was 2.0. The acidified mixture was stirred for a further 10mins. before separating the phases by centrifugation. The aqueous phasewas discarded. To the n-butanol extract 0.5% of Norit GSX carbon wasadded and the mixture stirred for 15 minutes. The carbon was discardedafter removal by filtration using a diatomaceous earth as a filter aid.To the n-butanol a 1/4 volume of deionized water was added and themixture stirred while adding 20% NaOH solution until the pH hadequilibrated a 7.0. The phases were separated by centrifugation and then-butanol phase discarded. The aqueous phase was concentrated underreduced vacuum to 800 ml. and then freeze-dried. This yielded 35g. of acrude solid preparation of clavulanic acid with an I₅₀ of 1.3 μg/ml inthe β-lactamase inhibition assay. This solid preparation was stored dryat -20° C while awaiting further purification.

EXAMPLE 15 Isolation of Crude Clavulanic Acid Sodium Salt

One liter of culture filtrate giving 53% inhibition at 1/2500 in theβ-lactamase inhibition assay and obtained as described in Example 12 waspercolated down a 1 inch diameter × 6 inch column of Permutit Isoporeresin FF 1P (SRA 62) in the Cl.sup. - form [supplied by Permutit Co.Ltd., 632-652 London Road, Isleworth, Middlesex, U.K.]. The culturefiltrate was followed by 300 ml. of distilled water to wash the column.Elution of the active β-lactamase inhibitor was achieved with 0.2M NaClsolution. Fractions (20 ml.) were collected and assayed at a 1/2500final dilution in the β-lactamase inhibition assay. Active fractionswere combined and concentrated under vacuum to 20 ml. This solution wasdesalted by gel exclusion chromatography on a Biorad Biogel P2 column11/2 inches in diameter with a gel bed of 16 inches and eluted with 1%n-butanol in water. [Biogel P2 is supplied by Bio Rad Laboratories, 32ndand Griffin Ave., Richmond, Calif., U.S.A.]. The active fractions, asdetermined by the β-lactamase inhibition assay, were combined. Sodiumchloride eluted after clavulanic acid and was detected using silvernitrate solution. The combined active fractions were concentrated andfreeze dried.

One liter of culture filtrate after the above treatment yielded 0.45g.of a crude solid preparation of clavulanic acid having an I₅₀ of 0.92μg/ml.

This solid was stored at -20° C while awaiting further purification.

EXAMPLE 16 Isolation of Crude Clavulanic Sodium Salt

Culture filtrate containing 300 μg/ml of clavulanic acid is acidifiedusing an in-line mixer system, extracted with n-butanol and clavulanicacid is back extracted into water at neutral pH.

Chilled culture filtrate (5°-10° C) was pumped to an in-line mixer atthe inlet of which, enough 6% (v/v) nitric acid was added to maintain anoutlet pH of 2.0 ± 0.1. The acidified filtrate was passed at 4.1/minthrough a glycol-cooled-plate heat exchanger (A.P.V. Ltd.) to maintain atemperature between 2°-5°. The pH was monitored in a flow-cell beforepassing into a three-stage counter-current separator (WestfaliaSeparator Ltd., Model EG 1006).

Chilled water saturated n-butanol (at about 5° C) was pumped at 3 l/mininto the counter-current separator.

The aqueous outlet from the counter-current separator was run to waste.Entrained water was removed from the butanol outflow of the countercurrent separator using a liquid/liquid centrifugal separator. (AlfaLaval Ltd. Model 3024X-G). The butanol was collected in a stainlesssteel vessel fitted with a cooling jacket in which it was stored atabout 5° C.

From the vessel, 40 l aliquots were removed and thoroughly mixed with 2l of chilled water (5° C), saturated with n-butanol. The pH of thismixture was adjusted to pH 6.8 ± 0.1 using 20% sodium hydroxidesolution.

This aqueous extract/butanol mixture was fed to a liquid/liquidcentrifugal separator (Sharples Centrifuge Ltd. Model M35PY-5 PH) at apumped rate of 2 l/min.

From 1800 l of culture filtrate, 90 l of aqueous phase was recovered,containing 39% of the clavulanic acid present in the culture filtrate.

15 l of the aqueous extract was adjusted from 2%, to 8%, total solids bythe addition of 60 g sodium chloride per liter, and spray-dried(Anhydro, Copenhagen, Type Lab S 1). The conditions used were: Feed rate2 l/hr Atomizer voltage 170 v; Heater setting 6-7; Inlet temp., 150° C;Outlet temp., 80° C.

The dried product, total weight 1 kg., contained 62% of the clavulanicacid present in the feedstock.

The remaining 75 l of aqueous extract was concentrated byultrafiltration (De Danske Sukkerfabrikker. Laboratory Module, MembraneType 900). The operating procedure was to re-circulate the retentatefrom a stainless steel tank, fitted with a cooling system, with theoutlet valve set so as to give a differential pressure across the 40membranes of 25 atmospheres. The temperature was maintained at 2°-5° Cand the pH at 6.8 ± 0.1 by addition of 2N hydrochloric acid, asnecessary. The volume was reduced to 34 l which contained 72% of theclavulanic acid present in the feedstock.

The aqueous concentrate was stored at about 5° C, adjusted to 8% solids,and spray dried as above. The dried material contained 75% of theclavulanic acid present in the feedstock to the spray drier.

The total spray-dried product, from the 90 l of aqueous extractcontained 69.4 g of clavulanic acid which was 72% of the clavulanic acidin the spray-drying feedstock and 21% of the clavulanic acid present inthe 1800 l of culture filtrate.

EXAMPLE 17 Partial Purification of Crude Clavulanic Acid

Crude clavulanic acid preparations obtained as described in Example 15were purified by ion exchange chromatography. Eighteen grams of materialprepared as described in Example 15 having an I₅₀ value of 1.3 μg/ml(final concentration) were dissolved in 25 ml. of distilled water andapplied to a 11/2 inches × 16 inches bed of Permutit FF 1P (SRA 62)resin in the chloride form. The column was eluted with a sodium chloridegradient formed by gravity feeding 0.5M sodium chloride into a mixingreservoir containing 1 liter of distilled water which in turn fed thechromatographic column. 10 ml. cuts were collected and β-lactamaseinhibitory activity assayed using a 1/2500 dilution of the fractions.Activity was eluted after a main band of color between fractions 24 and30. The active fractions were combined and concentrated to 30 ml.

This solution was desalted using a 2 inches × 18 inches bed of BioradBiogel P2 and eluting with 1% n-butanol in water. The 20 ml. fractionswere assayed for clavulanic acid content using the 62-lactamaseinhibition assay. The fractions were also spotted onto paper strips andsprayed with either the Ehrlich or the triphenyltetrazolium sprayreagents described in Description 3. β-lactamase inhibitory activitycorrelated with the pink or red spots respectively produced by thesereagents. Active cuts were combined, exclusing those containing sodiumchloride and concentrated under vacuum to dryness. This yielded 520 mg.of partially purified clavulanic acid sodium salt with an I₅₀ of 0.2μg/ml in the standard β -lactamase inhibitor assay.

This layer chromatography (silica gel) of this clavulanic acidpreparation gave the following R_(f) values: n-butanol/ethanol/water4:1:5 v/v top phase R_(f) 0.37; n-butanol/acetic acid/water 12:3:5 v/vR_(f) 0.44; isopropanol/water 7:3 v/v R_(f) 0.78. The zones weredetected by spraying with Ehrlich's reagent. 6-Aminopenicillanic run asa marker and detected with the same spray had R_(f) values of 0.38; 0.39and 0.77 respectively.

EXAMPLE 18 Partial Purification of Clavulanic Acid Sodium Salt

Culture filtrate produced as described in Example 12 was solventextracted as in Example 14 to give a solid preparation which was furtherpurified by ion exchange chromatography using Whatman diethylminoethylcellulose DE 52. This solid (10g). was dissolved in 20 ml. of distilledwater and applied to a 11/2 inches × 20 inches column of DE 52 cellulosepreviously equilibrated with 0.01M sodium phosphate buffer pH 7.5. Thecolumn was eluted with a NaCl gradient. 0.1M NaCl in 0.01M sodiumphosphate buffer pH 7.5 was fed into a mixing chamber containing 1 literof 0.01M phosphate buffer pH 7.5 which in turn was connected to thecolumn. Fractions (10 ml.) were collected and these were assayed for β-lactamase inhibitory activity at a dilution of 1/2500. The fractionswere also examined for antibacterial activity by the hole-in-plate assaymethod using nutrient agar plates seeded with Klebsiella aerogenes. Thefractions having the highest β -lactamase inhibitory activity and givingzones of inhibition in the hole-in-plate assay were combined,concentrated and then desalted on a Biorad Biogel P2 column. Thesefractions were shown to contain clavulanic acid by paper and thin layerchromatography.

EXAMPLE 19 Isolation of Solid Clavulanic Acid Sodium Salt

A partially purified solid preparation of clavulanic acid (500 mg)prepared as in Example 17 was loaded onto a Whatman microgranular CC 31cellulose column with 1 inch × 20 inches bed size. The chromatographicsolvent was n-butanol/ethanol/water 4:1:5 v/v, top phase. The column wasrun at 4° C and 4 ml. fractions collected. Fractions were tested for thepresence of clavulanic acid by spotting onto filter paper and sprayingwith the Ehrlich (pink spot) or triphenyltetrazolium (red spot) sprayreagents. These spot tests were confirmed by β -lactamase inhibitionassays at a 1/1250 dilution. Active fractions were combined and driedunder vacuum on a rotary evaporator. The solid was dissolved in a smallvolume of distilled water and freeze dried. A white solid preparation ofthe sodium salt of clavulanic acid was obtained (40 mg) having an I₅₀ of0.08 μ g/ml in the β-lactamase inhibition assay.

EXAMPLE 20 Isolation of Solid Clavulanic Acid Sodium Salt

Concentrated back extract (6 l) from ultrafiltration in Example 16)containing 10 g of clavulanic acid as determined by the β -lactamaseinhibition assay of Description 1. This was percolated at 1 l/hr onto a2 inch × 24 inch column of Permutit Zerolit FF 1 P SRA 62 anion exchangeresin in the chloride form. The column was then washed with 2 l ofdeionized water prior to elution with a sodium chloride gradient. Thegradient was formed by a reservoir containing 4 l of 1.4 m NaCl feedinga stirred reservoir containing 4 l of 0.7 NaCl which in turn wasconnected to a stirred reservoir containing 4 l of deionized water whichwas connected via a pump to the column. The column was eluted at 2.5ml/min and 25 ml fractions collected. Fractions were assayed by the β-lactamase inhibition assay. Active fractions (nos. 140-230) werecombined and vacuum evaporated to near dryness. Ethanol (500 mls) wasthen added and the solid filtered off after vigorous shaking. Theethanol extract was then vacuum evaporated to dryness on a rotaryevaporator and redissolved in deionized water (40 mls). This was loadedonto a 4 inch × 24 inch column of Biorad Biogel P₂ and eluted with a 1%n-butanol solution. Fractions were collected (25 ml) and assayed forβ-lactamase inhibitory activity at a 1/2500 final dilution. Tests forsodium chloride content on 1/25 dilutions of the fractions were madeusing silver nitrate solution. Those fractions containing clavulanicacid free of sodium chloride were combined, concentrated by evaporationof the solvent under reduced pressure to 20 mls and then freeze dried.This yielded 4.8 g of the sodium salt of clavulanic acid. (I₅₀ about 006 μg/ml)

EXAMPLE 21 Preparation of an Ester of Clavulanic Acid (Methyl Ester)##STR13##

19.8 mg. of the sodium salt of clavulanic acid was dissolved in 0.5 ml.dry dimethylformamide and treated with 0.25 ml. methyl iodide. Afterstanding at room temperature for 1.5 hours under anhydrous conditions,the solvents were removed in vacuo. The residue was purified by P.L.C.on silica gel (Kieselgel 60F254 supplied by E. Merck, Darmstadt,Germany) eluting with ethyl acetate to give clavulanic acid methyl esteras a colourless oil (R_(f) 0.38; red color with triphenyltetrazoliumchloride spray) which has the following properties:

Analysis: Found: C, 50.49; H, 5.43; N, 6.29. C₉ H₁₁ NO₅ Requires: C50.70; H, 5.20; N, 6.57. λ max (Methanol): no absorption >215 nm. ν max(Film): 3300-3600 (Broad), 1800, 1750, 1695 cm⁻¹ Approximate 1st orderN.M.R. (CDCl₃): 2.49 (broad S, 1, exchanged with D₂ O), 3.05. (d, l, J =17.5 Hz), 3.54 (dd, l, J = 17.5 Hz, J₂ = 2.5 Hz), 3.84 (S, 3) 4.24 (d,2, J = 7 Hz), 4.93 (dt, l, J = 7 Hz, J₂ = 1.5 Hz), 5.07 (d, l, J = 1.5Hz), 5.72 (d, l, J = 2.5 Hz). Molecular weight (mass spectrum) :213.0635. Calculated for C₉ H₁₁ NO₅ : 213.0637

Thin layer chromatography of the methyl ester showed a single zone ineach of the following solvent systems; butanol/ethanol/water 4:1:5 v/vtop phase R_(f) 0.75; isopropanol/water, 7:3 v/v R_(f) 0.95;ethylacetate/ethylalcohol 8:2 v/v R_(f) 0.87. The zones were detected bybioautography using Klebsiella aerogenes with added benzylpenicillin(synergism system).

EXAMPLE 22 Preparation of an Ester of Clavulanic Acid (p-nitrobenzylester) ##STR14##

Treatment of the sodium salt of clavulanic acid with p-nitrobenzylbromide in dry DMF gave, after P.L.C., a colorless oil whichcrystallized from chloroform-ether to give to p-nitrobenzyl ester ofclavulanic acid as white feathery needles, m.p. 111°-112° C, which onrecrystallization had a mp of 117.5° -118° C.

EXAMPLE 23 Preparation of an Ester of Clavulanic Acid (Benzyl Ester)##STR15##

Impure 3-(β-hydroxyethylidine)-7-oxo-4-oxa-1-azabicyclo[3,2,0]heptane-2-carboxylic acid sodium salt (thought to be roughly 55mg. of pure material) in dry dimethylformamide (0.64 ml.) was treatedwith benzyl bromide (0.18 ml.). The solution was kept at roomtemperature (approx. 17°-18° C) for 3 hours under anhydrous conditions.The reaction mixture was fractionated on silica gel, eluting with ethylacetate, to give in substantially pure form the benzyl ester of3-(β-hydroxyethylidine)-7-oxo-4-oxa-1-azabicyclo[3,2,0]heptane-2-carboxylic acid 63 mg.) as a colorless oil. i.r. (film1800, 1745, 1695 cm⁻¹ ; n.m.r. (CDCl₃), 2.25 (s,l, exchangeable with D₂O), 3.05 (d,l, J = 17Hz), 3.51 (dd,l,J=17 Hz, J₂ = 2.5 Hz), 4.24 (d,2,J-7.5Hz), 4.92 (dt, l,J=7.5Hz, J₂ =1.5Hz), 5.15 (d,l,J=1.5Hz), 5.24(s,2), 5.71 (d,l,J=2.5 Hz), 7.45 δ (s,5).

EXAMPLE 24 Preparation of the Benzyl Ester of Clavulanic Acid from CrudeExtracts of the Fulture Filtrate of S. Clavuligerus

Culture filtrate 20 l. obtained as described in Example 10 was vacuumevaporated using a climbing film evaporator to 5 l. The concentrate wasthen freeze-dried using an Edwards E.F.6 shelf freeze-drier manufacturedby Edwards High Vacuum Ltd. The 300g. of solid so obtained container 3g. of sodium Clavulanic acid as determined by the enzyme inhibitionassay. The solid was suspended in 900 ml. of dry dimethylformamide and150 ml. of benzyl bromide was added. The mixture was stirred for 2 hoursat room temperature and then diluted with 1 l. of ethyl acetate. Thereaction mixture was filtered and the filtrate concentrated to as low avolume as was possible. The oily residue was extracted with a further 1l. of ethyl acetate and the extract filtered. The filtrate was againconcentrated and the resulting oily residue loaded onto a 3 inch × 14inch silica gel column (Biogel Biosil A 100 mesh) in cyclohexane. Thecolumn was eluted with cyclohexane to remove benzyl bromide and thesolvent was then changed to ethyl acetate and 20 ml. fractionscollected. Fractions were tested for the presence of the benzyl ester ofclavulanic acid by spotting onto glass backed silica gel t.l.c. plates(Merck precoated silica gel 60 F 254) and spraying with2,3,5-triphenyl-tetrazolium chloride (TTC) spray reagent.

EXAMPLE 25 Preparation of Clavulanic Acid Benzyl Ester

Spray dried solid (3.3 kg) containing 69.4 g of clavulanic acid asdetermined by enzyme inhibition assay was obtained as described inExample 16. The solid was slurried in 5.5 l. of dimethylformamide and500 mls. of benzyl bromide added. After stirring at room temperature for2 hours, 12 l. of ethyl acetate were added and the solids removed byfiltration. The filtrate was vacuum evaporated to an oily residue (212g). The residue was loaded onto a column containing a 4 inch × 13 inchbed of silica gel (Hopkins & Williams MFC) in cyclohexane. The columnwas eluted with 12 l. of cyclohexane to remove excess benzyl bromide.The eluent was then changed to ethyl acetate and 500 ml. fractionscollected. These were tested for benzyl clavulanate content by spottingonto silica gel t.l.c. plates (Merck precoated silica gel 60 F 254) andspraying with 2,3,5 triphenyltetrazolium chloride (TTC) spray reagent.Fractions giving intense red spots were further examined by t l c onsilica gel with chloroform/ethyl acetate 8:2 as the solvent and sprayingthe developed plates with T T C spray. Fractions 5-13 contained the bulkof the ester, and these were combined and vacuum concentrated to an oil(79.3 g). This preparation was then chromatographed on a 4 inch × 18inch column of silica gel (Merck silica gel H type 60) withchloroform/ethyl acetate 8:2 as the solvent. Fractions Fractions givingintense red spots with this reagent were further examined by t.l.c. onsilica gel plates using chloroform-ethyl acetate 8:2 as the solvent andspraying the developed plates with TTC spray. The benzyl ester ofclavulanic acid runs at R_(f) 0.31 at 22° C. Fractions containing thisester were combined and concentrated to 15 ml. and this solution wasfurther chromatographed on a 11/2 inch × 16 inch silica gel column(Merck silica gel H, type 60) with chloroform/ethyl acetate 8:2 as thesolvent. 15 ml. fractions were collected and tested for the benzyl esteras described above. Those fractions containing the ester wereconcentrated to 8 ml. and finally purified by column chromatography on a1 inch × 16 inch silica gel column (Merck silica gel H, type 60) withethyl acetate cyclohexane 8:2 as the solvent. Selected fractions werecombined and vacuum evaporated to give pure benzyl ester as an oil, 160mg. were selected as described above and yielded on concentration 45.9g.of oil which was of 62% purity as adjudged by NMR spectroscopy.

This product was finally chromatographed on a 23/4 inch × 18 inch columnof Sephadex LH 20 (Pharmacia) in cyclohexane/chloroform 1:1. Afterselection of fractions and concentration a colourless oil (27.6 g) wasobtained which proved to be 95% pure benzyl ester of clavulanic acid asdetermined by NMR spectroscopic examination.

EXAMPLE 26 Preparation of Clavulanic Acid Benzyl Ester

Culture filtrate (150 l) pH 7.0 contained 16.2 g. of clavulanic acid(sodium salt) as determined by the enzyme inhibition assay. Thisfiltrate was stirred with 5 kg. of Amberlyst A.26 anion exchange resinin the chloride form (Rohm & Haas Company, Philadelphia, USA) for 1 hourat room temperature. The resin was then filtered and the filtratereassayed, showing that 6.4 g of clavulanic acid had been removed. Theresin was washed with 20 l. of deionized water followed by 20 l. ofacetone and 10 l. of dimethyl formamide (DMF). After refiltering theresin was suspended in 2.3 l. of DMF/0.2 M NaI. To this was added 200mls. of benzyl bromide and the suspension stirred thoroughly. Afterstanding at room temperature for 16 hours, ethyl acetate (2 l) wasadded, and the resin then filtered, further washings (Ethyl acetate) ofthe resin were combined with the filtrate. The extract was thenconcentrated to a small volume and chromatographed on 3 inch × 18 inchsilica gel column (Merck silica gel H type 60) with ethylacetate/cyclohexane 8:2 as the solvent. Fractions containing benzylclavulanate were selected by spotting onto silica gel t.l.c plates andspraying with TTC reagent as described previously (Example 24). Thoseselected were concentrated to 20 mls and then chromatographed on a 11/2inch × 18 inch silica gel column (Merck silica gel H type 60) withchloroform/ethyl acetate 8:2 as the solvent. Selected fractions werecombined and evaporated to a colourless oil (440 mgs) which was 90%benzyl clavulanate as determined by NMR spectroscopy.

EXAMPLE 27 Preparation of the Benzyl Ester of Clavulanic Acid from CrudeExtracts of the Culture Filtrate of S. clavuligerus

An aliquot of aqueous back extract of the butanol extract of culturefiltrate obtained as described in Example 14 was freeze-dried using anEdwards chamber drier. A 24 g. portion of the solid obtained contained0.96 g. of sodium clavulanic acid as determined by the enzyme inhibitionassay. This solid was suspended in 75 ml. of dry dimethylformamide and75 ml. of benzyl bromide was added. The mixture was stirred for 2 hoursat room temperature. The suspension was then diluted with 500 ml. ofethyl acetate and the mixture filtered. The filtrate was concentrated toan oily residue on a vacuum rotary evaporator. This residue was loadedonto a 2 inch × 14 inch silica gel column (Biogel Biosil A.100 mesh) incyclohexane. Benzyl bromide was eluted from the column and then thesolvent was changed to ethyl acetate and 10 ml. fractions was collected.Fractions containing the benzyl ester of clavulanic acid were selectedas in Example 24. Further purification was also achieved as described inExample 24 by column chromatography. This process yielded 220 mg. ofpure benzyl ester.

EXAMPLE 28 Preparation of Clavulanic Acid Sodium Salt ##STR16##

Substantially pure benzyl clavulanate (281 mg) in ethanol (25 ml.)containing sodium hydrogen carbonate (82 mg.) was hydrogenated over 10%Pd/C (90 mg.) for 25 minutes at room temperature and atmosphericpressure. The catalyst was filtered off, washed with water and ethanol,and the combined filtrates evaporated under reduced pressure at roomtemperature. The residual semi-solid was triturated with acetone,filtered and washed with ether to yield sodium clavulanate (135 mg.)

EXAMPLE 29 Hydrolysis of Clavulanic Acid Methyl Ester to Clavulanic Acid

2.17 mg. of clavulanic acid ester was dissolved in 0.1 ml. methanol andtreated with 0.208 ml. sodium hydroxide solution (0.0482N). After 1 hourat room temperature, the reaction mixture contained several products.T.L.C. analysis indicated that one of the major components had an R_(f)identical to that of the sodium salt of clavulanic acid; color reactionsand biological assay were consistent with this component being thesodium salt of clavulanic acid.

Slow conversion of the ester to clavulanic acid was seen when 1 mg/ml.of the compound was incubated at 37° C in 0.05M phosphate buffer at pH7. The reaction was followed by paper chromatography (bioautographicsystem). Using the butanol/ethanol/water system to follow the reactionover a period of 2 hours the zone of the methyl ester at R_(f) 0.79decreased in size as the zone of clavulanic acid at R_(f) 0.12increased.

EXAMPLE 30 Antibacterial Spectrum of Clavulanic Acid

The antibacterial activity of clavulanic acid sodium salt against arange of bacteria was determined using the microtitre method. Serialdilutions of clavulanic acid sodium salt in Oxoid sensitivity test brothcontained in a microtitre plastic tray were inoculated with an overnightbroth culture of each organism so that the final dilution of theinoculum was 0.5 × 10⁻⁴. The cultures were incubated overnight and thepoints of bacterial growth recorded next morning by observing theturbidity of the culture. The results, expressed as approximate MICvalues (minimum inhibitory concentration μg/ml.) are recorded in Table 3which shows that the compound has a broad spectrum of antibacterialactivity.

                  TABLE 3                                                         ______________________________________                                        ANTIBACTERIAL SPECTRUM OF CLAVULANIC ACID SODIUM SALT                                             Minimum Inhibitory                                        Bacterial Strain    Concentration μg/ml.                                   ______________________________________                                        Staphylococcus aureus (Oxford H)                                                                  7.5                                                       Staphylococcus aureus (Russell)                                                                   7.5                                                       Bacillus subtilis   62                                                        Streptococcus faecalis                                                                            >500                                                      Streptococcus pyogenes CN 10                                                                      125                                                       Escherichia coli NCTC 10418                                                                       31                                                        Klebsiella aerogenes                                                                              31 - 62                                                   Klebsiella oxytocum 62                                                        Enterobacter aerogenes T 624                                                                      31                                                        Enterobacter cloacae                                                                              62                                                        Acinetobacter anitratus                                                                           125                                                       Providentia stuartii                                                                              125                                                       Serratia marcescens 125                                                       Proteus mirabilis C977                                                                            62                                                        Proteus vulgaris W090                                                                             31                                                        Salmonella typhimurium                                                                            31                                                        Shigella sonnei     62                                                        Pseudomonas aeruginosa A                                                                          500                                                       ______________________________________                                    

EXAMPLE 31 Examples of β-Lactamase Inhibition by Clavulanic Acid SodiumSalt

Clavulanic acid progressively and irreversibly inhibits the β-lactamaseof Escherichia coli. The method of Description 1 shows that the otherβ-lactamases shown in Table 4 are also inhibited by clavulanic acid.

                  TABLE 4                                                         ______________________________________                                        INHIBITION OF β-LACTAMASES BY CLAVULANIC ACID                                              Approximate                                                                   I.sub.50 Value Relative to                                  Source of β-lactamase                                                                      Escherichia coli JT 4 = 1                                   ______________________________________                                        Staphylococcus aureus                                                                           1.0                                                         (Russell)                                                                     Escherichia coli JT4                                                                            1.0                                                         Escherichia coli B11                                                                            2.0                                                         Klebsiella aerogenes A                                                                          0.6                                                         Pseudomonas aeruginosa 1822                                                                     5.0                                                         (R factor)                                                                    Pseudomonas dalgleish                                                                           0.1                                                         ______________________________________                                    

With penicillin G as substrate the I₅₀ of clavulanic acid sodium saltagainst the β-lactamase of Staph. aureus (Russell) is approximately 0.06μg/ml.

EXAMPLE 32 Examples of Activity of Clavulanic Acid Methyl Ester

Tests for antibacterial activity in broth showed clavulanic acid methylester to have broad spectrum activity but of a lower order than shown byclavulanic acid. It was not clear whether this activity was the activityof the compound itself or of clavulanic acid liberated by slow aqueoushydrolysis of the ester. Clavulanic acid methyl ester showed markedantibacterial synergism in combination with ampicillin or cephaloridineagainst bacteria resistant to these antibiotics. Thus, the minimuminhibitory concentration (M.I.C.) for ampicillin against Staphylococcusaureus (Russell) was reduced from 500 μg/ml. to <0.4 in the presence of1.0 μg/ml. clavulanic acid methyl ester. The M.I.C. for cephaloridinewas reduced from 1.5 μg/ml. to <0.03 μg/ml. in the presence of 1 μg/ml.of clavulanic acid methyl ester. The M.I.C. for ampicillin againstProteus mirabilis C889 was reduced from 500 μg/ml. to 31 μg/ml. in thepresence of 5 μg/ml. clavulanic acid methyl ester.

EXAMPLE 33 Preparation Pivaloyloxymethyl Clavulanate

To a stirred solution of bromomethyl pivalate (0.37g) in drydimethylformamide (5 ml) was added sodium clavulanate (0.49g). After 2hrs. at room temperature the reaction mixture was treated with ethylacetate (20 ml), cyclohexane (10 ml) and water (20 ml). The mixtureseparated into two layers and the non-aqueous layer was separated,washed with water (20 ml) and dried over sodium sulphate. The driedsolution was evaporated to leave the required product as a pale yellowoil. (500 mg). N.m.r. (CDCl₃), 1.26 (s,9), 3.13 (d,l,J=17 Hz), 3.62 (dd,l,J,=17Hz, J₁ =2.5Hz, 4.3(d,2,J=7.5Hz), 5.0 (dt, l,J=7.5Hz, J₂ =1.5Hz),5.16(d,l,J=1.5Hz), 5.79(d,l,J=2.5Hz), 5.92δ(s,2); i.r.(liquid film), νβ-lactam C.O 1800 cm⁻¹, ester C═O 1760 cm⁻¹.

EXAMPLE 34 Preparation of Clavulanic Acid Phthalide Esters

To a stirred solution of 3-bromophthalide (0.43g) in drydimethylformamide (5 ml) was added sodium clavulanate (0.5g) and thesolution was left at room temperature for 2 hours. The solution wastreated with ethyl acetate (20 ml), cyclohexane (10 ml) and water (30ml) and shaken thoroughly. The non-aqueous layer was washed with water(20 ml), dried (Na₂ SO₄) and evaporated to yield a pale yellow gum. Thetwo diastereomeric esters were separated using high pressure liquidchromatography on a 40 cm × 10 mm column of silica gel (Merckosorb SI60, 5 μ) eluting with ethyl acetate at a flow rate of 3 ml/min.

The first phthalide ester (retention time 7.15 min) crystallized fromethyl acetate as needles, mp 102°, and had the following i.r. (Nujolmull) ν β-lactam C═O 1790 cm⁻¹ ester C═O 1755 cm⁻¹ n.m.r. (CD₃ COCD₃):3.14 (d,l,J=17.5Hz), 3.76 (dd,l,J,=17.5Hz, J₂ =2.5Hz),4.25(d,2,J=7.5Hz), 5.0 (dt,l,J₁ =7.5Hz, J₂ =1.5Hz), 5.4 (s,l,J=1.5Hz),5.82 (d,l,J=2.5Hz), 7.7 (s,1), 8.06δ(m,4); M.wt (mass spectrometry:331.0696 corresponds to C₁₆ H₁₃ NO₇ (calc. 331.0692). The seconddiasterioisomer (retention time 8.85 min) had the following i.r.(CH₂ Cl₂solution) ν β-lactam C═O 1800 cm⁻¹, ester C═O 1780 cm⁻¹ ; nmr (CDCl₃)2.42 (broad, S,1, exchangeable with D₂ O), 3.12 (d,l,J=18 Hz), 3.60(dd,l,J₁ =18 Hz, J₂ =2.5Hz), 4.30 (d,2,J=7.5Hz), 5.0 (dt,l,J₁ =7.5 Hz,J₂ =1.5 Hz), 5.12 (d,l,J=1.5 Hz), 5.76 (d,l,J=2.5 Hz), 7.52 (S,1),7.85δ(m,4). ##STR17##

EXAMPLE 35 Preparation of Nonyl Clavulanate

Sodium clavulanate (44 mg) in dry dimethylformamide (2 ml) was treatedwith nonyl iodide (76 mg) and left at room temperature for 2 hours. Thesolution was evaporated and the residue fractionated on silica gel,eluting with ethyl acetate-hexane (2:1 to give the product as an oil;i.r.(film) 1800, 1745, 1690 cm⁻¹. M.wt. (mas spectrometry) = 325.1890which corresponds to C₁₇ H₂₇ NO₅. (calc. 325.1889).

EXAMPLE 36 Preparation of Clavulanic Acid

Benzyl clavulanate (100 mgs) in ethanol (5 ml) was hydrogenated over 10%Pd/C (30 mgs) for 45 minutes at ambient temperature and atmosphericpressure. The catalyst was filtered, washed with ethanol and thecombined filtrates were evaporated in vacuo to give clavulanic acid asan unstable, viscous oil (58 mgs). N.m.r. (C₅ D₅ N): 3.05(d,l,J= 18Hz),3.60(dd,l,J₁ =18Hz, J₂ =2.5Hz), 4.75(d,2,J=7.5Hz),5.58(t,l,J=7.5Hz),5.66 (S,l), 6.0δ(d,l,J=18Hz).

EXAMPLE 37 Preparation of Methyl Clavulanate

Clavulanic acid (130 mgs) in ethanol (10 ml) was treated with excessdiazomethane in ether. After 2 minutes at room temperature the reactionwas shown (t.l.c) to be complete. The solution was evaporated in vacuoand the residue purified by chromatography on silica gel, eluting withethyl acetate. The fractions containing methyl clavulanate were combinedand evaporated to give a clear oil (104 mgs).

EXAMPLE 38 Preparation of Methyl Clavulanate

Clavulanic acid (200 mgs) in acetonitrile (5 ml) was cooled and stirredat 0°. Methanol (0.5 ml) and then dicyclohexyldicarbodiimide (206 mg.)were added and the reaction mixture was stirred at room temperatureovernight. The suspension was filtered and the filtrate evaporated invacuo to give crude methyl clavulanate. The crude product was purifiedby chromatography on silica gel, eluting with ethyl acetate, to give aclear oil (140 mg).

EXAMPLE 39 Preparation of Phenyl Clavulanate

Clavulanic acid (100 mg) in acetonitrile (5 ml) was cooled and stirredat 0°. To the solution was added phenol (0.94g) anddicyclohexyldicarbodiimide(100 mg) and the reaction mixture was stirredat room temperature overnight. The suspension was filtered and thefiltrate evaporated. The residue was fractionated on silica gel, elutingwith ethyl acetate-hexane (1:1) to give phenyl clavulanate (70 mg). I.r(film) 1800, 1770, 1690 cm⁻¹. N.m.r. (CDCl₃) 2.18 (broad s,l), 3.06 (dd,l,J=17Hz,J₂ =0.9Hz), 3.54 (dd,l,J₁ =17Hz,J₂ =2.6Hz), 4.29 (d,2,J=7.5Hz),5.1(dt,l,J₁ =7.5Hz,J₂ =1.5Hz) 5.29 (d,l,J=1.5Hz),5.76(dd,l,J₁ =2.6Hz,J₂=0.9Hz), 7.35δ(m,5). M.wt. (mass spectrometry) = 275.0777 whichcorresponds to C₁₄ H₁₃ NO₅ (calc. 275.0794).

EXAMPLE 40 Preparation of 2,2,2-trichloroethyl clavulanate

Sodium clavulanate (221 mgs) was suspended in dry tetrohydrofuran (5mls) and stirred at 0°. Trichloroethylchloroformate (211 mg) in drytetrohydrofuran (1 ml) was added to the above suspension over 20minutes. The mixture was allowed to reach room temperature and stirredovernight. The suspension was filtered and the filtrate evaporated invacuo. The residue was chromatographed on silica gel eluting with ethylacetate-hexane (2:1) to give the required product as an oil. i.r (film)1800,1760,1690 cm⁻¹. n.m.r. (CDCl₃) 1.56 (broad S,l), 3.07 (dd,l,J₁=17.5Hz,J₂ =0.7Hz), 3.56 (dd,l,J₁ =17.5Hz, J₂ =2.5Hz), 4.24(d,2,J=7.5Hz), 4.69 (d,l,J=12Hz), 4.92 (d,l,J=12Hz), 5.02 (dt,l,J₁=7.5Hz, J₂ =1.3Hz), 5.19(d,l,J=1.3Hz), 5.73 δ (dd,l,J₁ =2.5Hz, J₂=0.7Hz). M.wt. (mass spectrometry) = 328.9621 which corresponds to C₁₀H₁₀ NO₅ Cl₃ (calculated 328.9625). ##STR18##

EXAMPLE 41 Preparation of Sodium Clavulanate

Benzyl clavulanate (840 mgs) in ethanol (30 ml) and water (5 ml) washydrogenated over 10% Pd/C (267 mgs) and sodium bicarbonate (244 mgs)for 25 minutes at room temperature and atmospheric pressure. Thecatalyst was filtered, washed with water and ethanol and the combinedfiltrates were evaporated in vacuo. The product crystallized from awater-acetone mixture as microneedles (565 mgs). Recrystallization fromwater-acetone gave needles which, after drying over P₂ O₅ in vacuo for24 hours gave the following analysis: C. 41.01, 40,86; H 3.77, 3.64; N5.68, 5.51; i.r.(KBr disc) 1785, 1700, 1620 cm⁻¹ ; Nmr (D₂ O) 3.06(d,l,J=18.5Hz), 3.57 (dd,l,J₁ =18.5Hz, J₂ =2.5Hz), 4.15 (d,2,J=8Hz), 5.3(HOD), 4.9(m), 5.71 (d,l,J=2.5Hz)

EXAMPLE 42 Antibacterial, Synergism Between Amplicillin and ClavulanicAcid Sodium Salt

The minimum inhibitory concentration (M.I.C. values) of ampicillin,clavulanic acid sodium salt and ampicillin in the presence of 1 μg/ml.clavulanic acid sodium salt were determined for a range of β-lactamaseproducing bacteria. The organisms were inoculated into Oxoid sensitivitytest broth located in small wells in a plastic tray and containingseparate concentration gradients of ampicillin, clavulanic acid sodiumsalt or ampicillin plus 1 μg/ml. clavulanic acid sodium salt (microtitremethod). The final dilution of the inoculum was 0.5 ×10⁻². The tray wasincubated at 37° C overnight and a record made next morning of the endpoints of bacterial growth. The M.I.C. values in μg/ml. are recorded inTable 5 which reveals that the synergist at the low concentration of 1μg/ml. markedly enhances the antibacterial activity of ampicillinagainst certain Gram negative and Gram positive bacteria. The mechanismof this synergism is likely to involve inhibition of ampicillindestroying β-lactamase enzymes but the existence of other mechanismscannot be excluded.

Similar results to those shown in Table 5 were obtained when ampicillinwas replaced by amoxycillin or by the phthalidyl ester of ampicillin.

                  TABLE 5                                                         ______________________________________                                        ANTIBACTERIAL SYNERGISM BETWEEN AMPICILLIN AND                                CLAVULANIC ACID SODIUM SALT                                                             Minimum Inhibitory Concentrations μg/ml                                                          Ampicillin in                                             Clavulanic          presence of                                               acid                1μg/ml clavu-                                          sodium              lanic acid                                    Bacterial strain                                                                          salt      Ampicillin                                                                              sodium salt                                   ______________________________________                                        Escherichia coli                                                              NCTC 10481  31        1.8       <0.4                                          Escherichia coli                                                              B 11        62        >500      125                                           Klebsiella                                                                    aerogenes A 31        125       <0.4                                          Klebsiella sp 62                                                                          31        125       <0.4                                          Enterobacter                                                                  cloacae     62        250       62                                            Serratia                                                                      marcescens  125       >500      62                                            Staphylococcus                                                                aureus (Russell)                                                                          15        500       <0.4                                          Staphylococcus                                                                aureus      62        250       7.5                                           ______________________________________                                    

EXAMPLE 43 Antibacterial Synergism between Cephaloridine and ClavulanicAcid Sodium Salt

The minimum inhibitory concentrations of cephaloridine, clavulanic acidsodium salt and cephaloridine in the presence of 5 μg/ml clavulanic acidsodium salt were determined by the method described in Example 42. Theresults in Table 6 show that synergism can be obtained betweenclavulanic acid sodium salt and cephaloridine particularly for theβ-lactamase producing strain of Staphylococcus aureus (Russell).

                  TABLE 6                                                         ______________________________________                                        ANTIBACTERIAL SYNERGISM BETWEEN CEPHALORIDINE                                 AND CLAVULANIC ACID SODIUM SALT                                                        Minimum Inhibitory Concentrations μg/ml.                                     Clavulanic         Cephaloridine in                                           acid               presence of                                     Bacterial  sodium    Cephal-  5μg/ml clavulanic                            strain     salt      oridine  acid sodium salt                                ______________________________________                                        Proteus                                                                       mirabilis                                                                     899        >500*     62       7.5                                             Staphylococcus                                                                aureus (Russell)                                                                         15        3.1      <0.03.sup.+                                     Staphylococcus                                                                aureus     62        15       3.7                                             ______________________________________                                         *Tailing Point                                                                .sup.+ Same value obtained when synergist added at 1 μg/ml. instead of     5 μg/ml.                                                              

EXAMPLE 44 Antibacterial Synergism between Clavulanic Acid Sodium Saltand Various Penicillins

The results presented in Table 7 were obtained by the method describedin Example 42.

                  TABLE 7                                                         ______________________________________                                        ANTIBACTERIAL SYNERGISM BETWEEN CLAVULANIC ACID                               SODIUM SALT AND VARIOUS PENICILLINS AGAINST                                   STRAINS OF KLEBSIELLA AEROGENES                                               Amoxycillin    Carbenicillin*                                                                             Benzylpenicillin                                                +5μg/ml.    +5μg/ml.  +5μg/ml.                         Strain                                                                              Alone   synergist                                                                              Alone synergist                                                                            Alone synergist                           ______________________________________                                        A     500     0.97     500   7.8    250   7.8                                 E 70  500     3.9      500   15     500   15.6                                62    250     15.6     125   7.8    250   15.6                                ______________________________________                                         *Similar results observed when carbenicillin replaced by carbenicillin        phenyl α-ester or ticacillin.                                      

EXAMPLE 45 Antibacterial Synergism between Ampicillin and Esters ofClavulanic Acid

The results presented in Table 8 were obtained by the method describedin Example 42

                  TABLE 8                                                         ______________________________________                                        ANTIBACTERIAL SYNERGISM BETWEEN AMPICILLIN AND                                ESTERS OF CLAVULANIC ACID AGAINST STRAINS OF                                  KLEBSIELLA AEROGENES                                                                            Ampicillin   Ampicillin                                                       5 μg/ml of                                                                              5 μg/ml of                                                    Methyl Ester Benzyl Ester                                                     of           of                                                    Ampicillin clavulanic   clavulanic                                     Strain Alone      acid         acid                                           ______________________________________                                        A      500        1.9          1.9                                            E 70   500        3.9          3.9                                            62     500        3.9          3.9                                            ______________________________________                                    

Neither clavulanic acid methyl ester nor clavulanic acid benzyl esterinhibited the growth of the test organisms at a concentration of 100μg/ml.

EXAMPLE 46 ANTIBACTERIAL ACTIVITY OF CLAVULANIC ACID ESTER

The method of Example 30 but using a dilution of 1/100 of overnightbroth, the MIC values in Table 9 were obtained for certain esters ofclavulanic acid against a number of organisms:

                  TABLE 9                                                         ______________________________________                                        ANTIBACTERIAL ACTIVITY OF CLAVULANIC ACID ESTERS                                     MIC of Ester of Clavulanic Acid                                                                   MIC*                                                                        Pivalo-       of                                                              yloxy-        clavulanic                                      Benzyl  Nonyl   methyl                                                                              Phthalidyl                                                                            acid sodium                            Organism ester   ester   ester ester   salt                                   ______________________________________                                        Bacillus                                                                      subtilis A                                                                             250     31      62    125     62                                     Staph. aureus                                                                 Oxford    62     31      31    31      15                                     Staph. aureus                                                                 Russell  125     31      62    15      15                                     Escherchia                                                                    coli 10418                                                                             125     250     125   125     125                                    ______________________________________                                         *The MIC of clavulanic acid sodium salt is included for comparison; the       high MIC values (if compared to those of Example 30) are due to the heavy     inocula used.                                                            

EXAMPLE 47 EXTRACTION OF CLAVULANIC ACID USING LIQUID ION EXCHANGE RESIN

Culture filtrate (200 ml, obtained in a similar manner to Example 3butusig a medium containing 0.1% v/v KH₂ PO₄ instead of 0.01% FeSO₄.7H₂O) was extracted with Amberlite* LA2⁺ (Cl⁻ form, 15% v/v inmethylisobutyl ketone, 66 ml) for 30 minutes at 5° C.

The phases were separated by centrifugation (1660 g, 20 minutes). Thesolvent phase (60 ml) was recovered by pipette and divided into fourequal portions. Each portion was extracted by stirring at 5° C for 20minutes with 1/4 volume (3.75 ml) aqueous extractant as indicated in thetable below. The resulting mixture was centrifuged (1660 g, 15 minutes).3.6 ml. aqueous phase was recovered from each extraction.

    ______________________________________                                                            Clavulanic acid                                                       Volume  concentration Clavulanic                                  Sample      (ml)    (μg ml.sup.-1)                                                                           acid (mg)                                   ______________________________________                                        clarified brew                                                                            200     128           25.4                                        extracted brew                                                                            200     15            3.0                                         M NaCl extract                                                                            3.6     305           1.1                                         2M NaCl extract                                                                           3.6     598           2.5                                         M NaNO.sub.3 extract                                                                      3.6     638           2.3                                         2M NaNO.sub.3 extract                                                                     3.6     758           2.73                                        ______________________________________                                    

The result obtained with 2M NaNO₃ represents a recovery of 43% fromclarified brew.

EXAMPLE 48 EXTRACTION OF CLAVULANIC ACID USING LIQUID ION EXCHANGE RESIN

Clarified brew (47 liters, obtained as in Example 12) was extracted withAmberlite LA2 (acetate form, 15% v/v in methylisobutyl ketone, 12.5liters) by stirring for 1 hour at 17° C. After adding octan-1-ol (500ml) the phases were separated in a continuous flow centrifuge yielding9.2 liters solvent phase, which was then stirred at 5° C for 11/2 hourswith molar sodium nitrate (2.3 liters). The mixture was separated bycontinuous flow centrifugation yielding 2.4 liters aqueous phase(including water used for displacement purposes). Aqueous phase pH(initially 8.0) was adjusted to 7.0 with concentrated hydrochloric acid.

    ______________________________________                                                             clavulanic acid                                                                             clavulanic                                             Volume   concentration acid                                       Sample      (1)      (μg ml.sup.-1)                                                                           (mg)                                       ______________________________________                                        clarified brew                                                                            47       146           6862                                       extracted brew                                                                            47       19             893                                       M NaNO.sub.3 extract                                                                      2.4      1638          3931                                       ______________________________________                                    

Extraction efficiency from clarified brew to sodium nitrate extract is57%. ##SPC1##

Clavulanic acid and its salts and esters are antibacterial agents whichact synergistically with penicillins and cephalosporins, possiblybecause of their ability to inhibit certain β-lactamases. It has nowbeen found that amides of clavulanic acid are also useful β-lactamaseinhibitors and that they can enhance the activity of penicillins andcephalosporins against certain bacteria.

Accordingly the present invention provide amides of the compound of theformula (I).

One particularly suitable group of compounds of this invention is thatof the formula (II): ##STR19## and their salts wherein R₁ is a hydrogenatom or an organic group of 1-7 carbon atoms and R₂ is a hydrogen atomor anorganic group of 1-7 carbon atoms.

Suitably R₁ is a hydrogen atom or a hydrocarbon group of 1-7 carbonatoms optionally substituted by halogen atoms or a methoxyl group.

Most suitably R₁ is a hydrogen atom or a C₁₋₄ alkyl group optionallysubstituted by halogen atoms or a phenyl, tolyl, or benzyl group.

Preferably R₁ is a CF₃ CH₂ -group.

Most suitably R₂ is a hydrogen atom or a C₁₋₄ alkyl group.

Preferably R₂ is a hydrogen atom.

A further particularly suitable group of compounds of this invention isthat of the formula (III): ##STR20## and their salts wherein R₃ is apenicillin or cephalosporin containing an amino-acyl side chain or anester thereof.

Suitable esters of the penicillins or cephalosporins include thoseesters notionally drived from an alcohol ROH where R is an organicresidue containing from 1 to 12 carbon atoms.

Suitably the ester will be hydrogenolysable ester, such as the benzylester, or hydrolysable ester, particularly an in-vivo hydrolysableester, such as the phthalidyl or pivaloyloxymethyl ester, of thepenicillin or cephalosporin.

Suitable penicillins and cephalosporins include ampicillin, amoxycillin,epicillin, cyclacillin, 6-aminopenicillanic acid, 7-aminocephalosporanicacid, 7-aminodesacetoxycephalosporanic acid, cephalexin, cephadine andcephaloglycine.

Preferably R₃ is the benzyl, phthalidyl or pivaloyloxymethyl ester ofampicillin or amoxycillin.

Other particularly suitable compounds of this invention include that ofthe formula (IV): ##STR21## wherein CO₂ R₄ is a carboxylic acid group ora salt or ester thereof.

Most suitably the salts of the compound of the formula (IV) will bepharmaceutically acceptable salts, such as the sodium, potassium,calcium, magnesium, aluminium and ammonium salts.

Preferred salts include the sodium and potassium salt.

Crystalline forms of salts may contain water of hydration.

Suitable esters of the compound of the formula (IV) include those estersof clavulanic acid described above.

Most suitably the ester of the compound of the formula (IV) will be ahydrogenolysable ester, such as the benzyl ester, or an in-vivohydrolysable ester, such as the phthalidyl or pivaloyloxymethyl ester.

The present invention also provides pharmaceutical compositions whichcomprise a compound of this invention together with a pharmaceuticallyacceptable carrier.

The compositions of the invention include those in a form adapted fororal, topical or parenteral use and may be used for the treatment ofinfections in mammals including humans.

Suitable forms of the compositions of this invention include tablets,capsules, creams, syrups, suspension, solutions, reconstituable powdersand sterile forms suitable for injection or infusion. Such compositionsmay contain conventional pharmaceutically acceptable materials such asdiluents, binders, colours, flavours, preservatives, disintegrants andthe like in accordance with conventional pharmaceutical practice in themanner well understood by those skilled in the art of formulatingantibiotics.

Injectable or infusable compositions of salts of a compound of thisinvention are particularly suitable as high tissue levels of a compoundof this invention can occur after administration by injection orinfusion. Thus, one preferred composition aspect of this inventioncomprises a salt of a compound of this invention in sterile form.

Unit dose compositions comprising a compound of this invention or a saltor ester thereof adapted for oral administration form a furtherpreferred composition aspect of this invention.

Under certain conditions, the effectiveness of oral compositions of acompound of this invention and its salts and esters can be improved ifsuch compositions contain a buffering agent or an enteric coating agentsuch that the compounds of the invention do not have prolonged contactwith highly acidic gastric juice. Such buffered or enterically coatedcompositions may be prepared in accordance with conventionalpharmaceutical practice.

The compound of this invention or its salt or ester may be present inthe composition as sole therapeutic agent or it may be present togetherwith other therapeutic agents such as a β-lactam antibiotic. Suitableβ-lactam antibiotics for inclusion in such synergistic compositionsinclude not only those known to be highly susceptible to β-lactamasesbut also those which have a good degree of intrinsic resistance toβ-lactamases. Thus, suitable β-lactam antibiotics for inclusion in thecompositions of this invention include benzylpenicillin,phenoxymethylpenicillin, carbenicillin, methicillin, propicillin,ampicillin, amoxycillin, epicillin, ticarcillin, cyclacillin,6-aminopenicillanic acid, 7-aminocephalosporanic acid,7-aminodesacetoxycephalosporanic acid, cephaloridine, cephalothin,cefazolin, cephalexin, cefoxitin, cephacetrile, cephamandole,cephapirin, cephradine, cephaloglycine and other well known penicillinsand cephalosoprins or pro-drugs therefore such as hetacillin,metampicillin, the acetoxymethyl, pivaloyloxymethyl or phthalidyl estersof benzyl penicillin, amipicillin, amoxycillin or cephaloglycine or thephenyl tolyl or indanyl α-esters of carbenicilin or ticarcillin or thelike.

Naturally if the penicillin or cephalosporin present in the compositionis not suitable for oral administration then the composition will beadapted for parenteral administration.

When present in a pharmaceutical composition together with a β-lactamantibiotic, the ratio of a compound of the formula (II) or its salt orester present to β-lactam antibiotic present may be from, over a widerange of ratios, for example, 10:1 to 1:10 and advantageously may befrom 3:1 to 1:3, for example, 1:1.

The total quantity of antibacterial agents present in any unit dosageform will normally be between 50 and 1500mg and will usually be between100 and 1000mg.

Compositions of this invention may be used for the treatment ofinfections of inter alia, the respiratory tract, the urinary tract andsoft tissues in humans.

Compositions of this invention may also be used to treat infections ofdomestic animals such as mastitis in cattle.

Normally between 50 and 3000mg of the compounds of the invention will beadministered each day of treatment but more usually between 100 and1000mg of the compounds of the invention will be administered per day.However, for the treatment of severe systemic infections or infectionsof particularly intransigent organisms, higher doses may be used inaccordance with clinical practice.

The exact form of the compositions of this invention will depend to someextent on the micro-organism which is being treated. For treatment ofinfections the compositions of this invention are normally adapted tocontain at least 0.25μg/ml, and preferably at least 1μg/ml of a compoundof this invention.

The penicillin or cephalosporin in synergistic compositions of thisinvention will normally be present by up to or at approximately theamount conventionally used when that penicillin or cephalosporin is thesold therapeutic agent used in the treatment of infection.

Particularly favoured compositions of this invention will contain from150 - 1000mg of amoxycillin, ampicillin or a pro-drug thereof and from50 - 500mg of a compound of this invention or a salt or in-vivohydrolysable ester thereof and more suitable from 200 - 500mg ofamoxycillin, ampicillin or a pro-drug therefore and from 50 - 250mg of acompound of this invention or a salt or in vivo hydrolysable esterthereof.

The materials present in such compositions may be hydrated if required.The weights of the antibotics in such compositions are expressed on thebasis of antibiotic theoretically available from the composition and noton the basis of the weight of pro-drug.

The compounds of the present invention may be prepared by the reactionof the compound of the formula (I) with an amine in the presence of acondensation promoting agent.

The compounds of the present invention may also be prepared by thereaction of a salt of the compound of the formula (I) with a salt of anamine in the presence of a condensation promoting aent.

Suitable salts of the compound of the formula (I) are the calcium,sodium and potassium salts. Suitable salts of the amine include thehydrobromide, hydrochloride and tosylate salts.

Suitable condensation promoting agents include carbodiimides such asdicyclohexylcarbodiimide, 1-cyclohexy-3-(2'-morpholinoethyl)carbodiimide and the like.

Most suitably the reactions are carried out at a non-extremetemperature, for example -30° to +80° C and conveniently at ambienttemperature.

These reactions are carried out in a solvent inert under the reactionconditions such as water, dioxane, tetrahydrofuran and the like ormixtures of such solvents.

The reaction will preferably be carried out by the reaction ofclavulanic acid with an amine HNR₂ R₂, R₃ NH₂ or NH₂ CH₂ CO₂ R₄, whereinR₁, R₂ and R₃ are as hereinbefore defined and the CO₂ R₄ group is anester group as hereinbefore defined, in an aqueous solvent, for exampledioxan-water or tetrahydrofuran-water.

The carboxylic acid function in the compound of the formula (IV) may beconverted to another carboxylic acid function by conventional methodswell known to those skilled in the art. Belgian Pat. No. 827,926describes particularly suitable methods for performing such conversions.

EXAMPLE 49 ##STR22##

A solution of clavulanic acid (1) was prepared by hydrogenation ofbenzyl clavulanate (2.89g; 0.01 mole) in redistilled tetrahydrofuran(30ml) over 10% palladised charcoal (0.8g) for 20 min. at 22°. Afterfiltering to remove the catalyst, an aliquot of the solution wastitrated to pH 7 with N/1 sodium hydroxide solution to confirm that itwas 0.3M (Solution A).

Solution A (14ml; 4 m. moles) and a 0.28 M solution of ammonia intetrahydrofuran (15ml; 4 m. moles) were added simultaneously withstirring to a solution of dicyclohexyl carbodiimide (0.75g; < 4 m.moles) in dichloromethane (100ml). There was an immediate whiteprecipitate. The mixture was evaporated to dryness under reducedpressure and the residue treated with dichloromethane (20ml). After 2hrs. at room temperature the solvents were evaporated and the residuepartitioned between ethyl acetate (20 ml), ether (10ml) and water(20ml). The insoluble material was filtered off, the filtrate separated,and the aqueous layer evaporated to dryness in vacuo, to yield 0.6g(75%) of crude clavulanamide (2) as a pale yellow crystalline solid. Itwas crystallised by dissolution in warm methanol (10ml), addition ofethyl acetate (10ml) and filtration, followed by the evaporation off ofmost of the methanol under reduced pressure. After cooling to 2°-3° Cthe product (0.15g) was collected, washed with methanol-ethyl acetate,then with ether, and dried, m.p. 166° C.

EXAMPLE 50 ##STR23##

Solution A (14ml; 4 m. moles) and solutions of benzylamine (0.4g; 4 m.moles) in ethyl acetate (100ml) and dicyclohexyl carbodiimide (0.8g; 4m. moles) in dichloromethane were mixed quickly together. The mixturewas evaporated to dryness and dissolved as far as possible indichloromethane (100ml). It was allowed to stand at room temperature for1 hour, cooled to 2°-3°, filtered and re-evaporated. The residue wastriturated with ether-ethyl acetate and the crude product (3) collectedand dried (0.8g; 78%) m.p. 131°. After crystallization from ethylacetate it had m.p. 139°.

EXAMPLE 51 ##STR24##

Solutions of sodium clavulanate tetrahydrate (0.3g; 1m. mole) in water(2ml), 2,2,2-trifluoroethylamine hydrochloride (0.14g; 1 m. mole) inwater (2ml) and1-cyclohexyl-3-(2'-N-morpholinoethyl)carbodiimide-metho-p-toluenesulphonate(0.43g; 1m. mole) in water (2ml) and dioxan (1ml) were mixed and stirredat room temperature for 1/2 hour. The mixture was evaporated to drynessunder reduced pressure, and the residue treated with ethyl acetate(25ml) and anhydrous sodium sulphate (5g). The ethyl acetate solutionwas filtered and evaporated to a crystalline residue which wastriturated with ether and then with a little dichloromethane to give theamide (5) as a colourless crystalline solid, m.p. 168°-72° (sublimes),50mg.

EXAMPLE 52 ##STR25##

Solution A (3ml; 1 m. mole) glycine benzyl ester (free base from 0.34g;1 m. mole of p. toluenesulphonate salt) in ethyl acetate (20ml) anddicyclohexylcarbodiimide (0.2g; 1 m. mole) in dichloromethane (20ml)were mixed and allowed to stand for 1 hour at room temperature. Themixture was cooled in ice-water, filtered to remove the urea, andevaporated to dryness under reduced pressure.

The residue was purified by column chromatography on silica gel usingethyl acetate as eluent, to give the amide (6) which aftercrystallization from ether-ethyl acetate had m.p. 120°.

Using a strictly analogous method the amides formed frompivaloyloxymethyl 6-aminopenicillanate (7) and the phthalide ester ofampicillin were prepared (8).

EXAMPLE 53 ##STR26##

Solutions of sodium clavulanate (4) (150mg) in water (1ml), p-tolinehydrochloride (80g) in water (1ml) and 1-cyclohexyl-3-(2'morpholinomethyl) carbodiimide metho-p-toluenesulphonate (210mg) in 1:2dioxan-water (3ml) were mixed and stirred at room temperature. After 20minutes the precipitated toluidide was collected, washed with water anddried in vacuo to give the amide (9) (95mg) m.p. 223° C.

EXAMPLE 54 ##STR27##

Solutions of: sodium clavulanate (4) (150mg) in water (1ml), benzyl6-aminopenicillanate p.toluenesulphonate (240mg) in 1:2 dioxan-water (3ml) and 1-cyclohexyl-3-(2'morpholinoethyl)-carbodiimide (210mg) in 1:2dioxan-water (3ml) were mixed and stirred for 25 minutes. The solutionwas extracted with ether-ethylacetate (1:1, 25ml) separated, and thenon-aqueous layer washed successively with 5% aqueous p.toluenesulphonic acid (2 × 10ml), 3% aqueous sodium hydrogen carbonate(10ml) and water (10ml). The solution was dried over anhydrous sodiumsulphate and the solvent was evaporated in vacuo to give the amide (10)as a gum (50mg) I.r. (liquid film) ν C═O 1797 cm⁻¹ (clavam ⊕-lactam) νC═O 1785 cm⁻¹ (penam β-lactam) ν C═O 1745 cm⁻¹ (ester carbonyl) ν max1695 cm⁻¹ (amide C═O and C═C).

What we claim is:
 1. A compound of the formula (II): ##STR28## wherein R₁ is hydrogen, alkyl of 1 to 4 carbon atoms, CF₃ CH₂, phenyl, tolyl or benzyl and R₂ is hydrogen or alkyl of 1 to 4 carbon atoms.
 2. A compound according to claim 1 wherein R₁ is CF₃ CH₂.
 3. A compound according to claim 1 wherein R₂ is hydrogen.
 4. The compound according to claim 1 which is ##STR29##
 5. The compound according to claim 1 which is ##STR30##
 6. The compound according to claim 1 which is ##STR31##
 7. The compound according to claim 1 which is ##STR32## 